Detection of mutations in a gene associated with resistance to viral infection, oas1

ABSTRACT

A method for detecting a mutation related to the gene encoding OAS1. This and other disclosed mutations correlate with resistance of humans to viral infection including hepatitis C. Also provided is a therapeutic agent consisting of a protein or polypeptide encoded by the mutated gene, or a polynucleotide encoding the protein or polypeptide. Inhibitors of human OAS1, including antisense oligonucleotides, methods, and compositions specific for human OAS1, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.10/972,135 entitled Detection of Mutations in a Gene Associated WithResistance to Viral Infection, OAS1 filed Oct. 22, 2004, and claims thebenefit of Provisional U.S. Patent Application Nos. 60/513,888 filedOct. 23, 2003, 60/542,373 filed Feb. 6, 2004, 60/554,758 filed Mar. 19,2004, 60/560,524 filed Apr. 8, 2004, 60/578,323 filed Jun. 9, 2004, and60/605,243 filed Aug. 26, 2004, and are incorporated in their entiretyherein by reference.

This invention was made with government support under grant numberAI055137 awarded by The National Institute of Allergy and InfectiousDisease, and grant number CA90095 awarded by the National CancerInstitute. The Government has certain rights in the invention.

1. TECHNICAL FIELD

The present invention relates to a method for detecting a mutation in ahuman oligoadenylate synthetase gene, wherein a mutation confersresistance to flavivirus infection, including infection by hepatitis Cvirus, and a mutation relates to other disease states including prostatecancer and diabetes, and uses of the encoded proteins and antibodiesthereto.

2. BACKGROUND OF THE INVENTION

A number of diseases have been identified to date in which naturalresistance to infection exists in the human population. Alter and Moyer,J. Acquir. Immune Defic. Syndr. Hum Retrovirol. 18 Suppl. 1:S6-10 (1998)report hepatitis C viral infection (HCV) rates as high as 90% inhigh-risk groups such as injecting drug users. However, the mechanism bywhich the remaining 10% are apparently resistant to infection has notbeen identified in the literature. Proteins that play a role in HCVinfection include the 2-prime, 5-prime oligoadenylate synthetases. OASsare interferon-induced proteins characterized by their capacity tocatalyze the synthesis of 2-prime,5-prime oligomers of adenosine(2-5As). Hovanessian et al., EMBO 6: 1273-1280 (1987) found thatinterferon-treated human cells contain several OASs corresponding toproteins of 40 (OAS1), 46 (OAS1), 69, and 100 kD. Marie et al., Biochem.Biophys. Res. Commun. 160:580-587 (1989) generated highly specificpolyclonal antibodies against p69, the 69-kD OAS. By screening aninterferon-treated human cell expression library with the anti-p69antibodies, Marie and Hovanessian, J. Biol. Chem. 267: 9933-9939 (1992)isolated a partial OAS2 cDNA. They screened additional libraries withthe partial cDNA and recovered cDNAs encoding two OAS2 isoforms. Thesmaller isoform is encoded by two mRNAs that differ in the length of the3-prime untranslated region.

Northern blot analysis revealed that OAS2 is expressed as fourinterferon-induced mRNAs in human cells. The predicted OAS2 proteinshave a common 683-amino acid sequence and different 3-prime termini.According to SDS-PAGE of in vitro transcription/translation products,two isoforms have molecular masses of 69 and 71 kD. Both isoformsexhibited OAS activity in vitro. Sequence analysis indicated that OAS2contains two OAS1-homologous domains separated by a proline-richputative linker region. The N- and C-terminal domains are 41% and 53%identical to OAS1, respectively.

By fluorescence in situ hybridization and by inclusion within mappedclones, Hovanian et al., Genomics 52: 267-277 (1998) determined that theOAS1, OAS2, and OAS3 genes are clustered with a 130-kb region on12q24.2. 2-5As bind to and activate RNase I, which degrades viral andcellular RNAs, leading to inhibition of cellular protein synthesis andimpairment of viral replication.

A fourth human OAS gene, referred to as OASL, differs from OAS1, OAS2and OAS3 in that OASL lacks enzyme activity. The OASL gene encodes atwo-domain protein composed of an OAS unit fused to a 164 amino acidC-terminal domain that is homologous to a tandem repeat of ubiquitin.(Eskildsen et al., Nuc. Acids Res. 31:3166-3173, 2003; Kakuta et al., J.Interferon & Cytokine Res. 22:981-993, 2002.)

Because of their role in inhibiting viral replication and viralinfection, there is a need in the art for methods and compositions thatsuppress viral replication related to OAS1 activity, including aprofound need for inhibitor-based therapies that suppress HCVreplication.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to detecting hepatitis Cresistance-related mutations which are characterized as point mutationsin the oligoadenylate synthetase 1 gene.

In one embodiment, a human genetic screening method is contemplated. Themethod comprises assaying a nucleic acid sample isolated from a humanfor the presence of an oligoadenylate synthetase 1 gene point mutationcharacterized as a base substitution at nucleotide position 2135728,2135749, 2135978, 2144072, 2144088, 2144116, 2144321, 2131025, 2133961,2139587, 2144294, 2144985, 2156523, or 2156638 or a base deletion atnucleotide position 2156595 for oligoadenylate synthetase 1 gene (OAS1)with reference to Genbank Sequence Accession No. NT_(—)009775.13(consecutive nucleotides 2,130,000-2,157,999 of which are shown in FIG.2 as SEQ ID NO:19).

In a preferred embodiment, the method comprises treating, underamplification conditions, a sample of genomic DNA from a human with apolymerase chain reaction (PCR) primer pair for amplifying a region ofhuman genomic DNA containing nucleotide position 2135728, 2135749,2135978, 2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587,2144294, 2144985, 2156523, 2156595, or 2156638 of oligoadenylatesynthetase 1 gene NT_(—)009775.13. The PCR treatment produces anamplification product containing the region, which is then assayed forthe presence of a point mutation.

In a further embodiment, the invention provides a protein encoded by agene having at least one mutation at position 2135728, 2135749, 2135978,2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294,2144985, 2156523, 2156595, or 2156638 of NT_(—)009775.13, and use of theprotein to prepare a diagnostic for resistance to viral infection,preferably flaviviral infection, most preferably hepatitis C infection.In specific embodiments, the diagnostic is an antibody.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or inhibiting infection by a virus, preferably aflavivirus, most preferably hepatitis C virus, wherein the therapeuticcompound is a protein encoded by an OAS1 gene having at least onemutation at position 2135728, 2135749, 2135978, 2144072, 2144088,2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985, 2156523,2156595, or 2156638 of NT_(—)009775.13. In other embodiments thetherapeutic compound is a polynucleotide, such as DNA or RNA, encodingthe protein.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or inhibiting infection by a virus, preferably aflavivirus, most preferably a hepatitis C virus, wherein the therapeuticcompound is a protein of the sequence: SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:47 and/or SEQ IDNO:48.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or inhibiting infection by a virus, preferably aflavivirus, most preferably hepatitis C virus, wherein the therapeuticcompound mimics the beneficial effects of at least one mutation atposition 2135728, 2135749, 2135978, 2144072, 2144088, 2144116, 2144321,2131025, 2133961, 2139587, 2144294, 2144985, 2156523, 2156595, or2156638 of NT_(—)009775.13. The therapeutic compound can be a smallmolecule, protein, peptide, DNA or RNA molecule, or antibody.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or treating cancer, preferably prostate cancer,wherein the therapeutic compound is a protein encoded by an OAS1 genehaving at least one mutation at position 2135728, 2135749, 2135978,2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294,2144985, 2156523, 2156595, or 2156638 of NT_(—)009775.13. In otherembodiments the therapeutic compound is a polynucleotide, such as DNA orRNA, encoding the protein.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or treating cancer, preferably prostate cancer,wherein the therapeutic compound is a protein of the sequence: SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:46,SEQ ID NO:47 and/or SEQ ID NO:48.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or treating cancer, preferably prostate cancer,wherein the therapeutic compound mimics the beneficial effects of atleast one mutation at position 2135728, 2135749, 2135978, 2144072,2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985,2156523, 2156595, or 2156638 of NT_(—)009775.13. The therapeuticcompound can be a small molecule, protein, peptide, DNA or RNA molecule,or antibody.

In further embodiments, the therapeutic compound is capable ofinhibiting the activity of OAS1 or at least one sub-region orsub-function of the entire protein, and such compounds are representedby antisense molecules, ribozymes, and RNAi molecules capable ofspecifically binding to OAS1 polynucleotides, and by antibodies andfragments thereof capable of specifically binding to OAS1 proteins andpolypeptides.

The present invention provides, in another embodiment, inhibitors ofOAS1. Inventive inhibitors include, but are not limited to, antisensemolecules, ribozymes, RNAi, antibodies or antibody fragments, proteinsor polypeptides as well as small molecules. Exemplary antisensemolecules comprise at least 10, 15 or 20 consecutive nucleotides of, orthat hybridize under stringent conditions to the polynucleotide of SEQID NO:19. More preferred are antisense molecules that comprise at least25 consecutive nucleotides of, or that hybridize under stringentconditions to the sequence of SEQ ID NO:19.

In a still further embodiment, inhibitors of OAS1 are envisioned thatspecifically bind to the region of the protein defined by thepolypeptide of SEQ ID NO:30. Inventive inhibitors include but are notlimited to antibodies, antibody fragments, small molecules, proteins, orpolypeptides.

In a still further embodiment, inhibitors of OAS1 are envisioned thatare comprised of antisense or RNAi molecules that specifically bind orhybridize to the polynucleotide of SEQ ID NO:31.

In further embodiments, compositions are provided that comprise one ormore OAS1 inhibitors in a pharmaceutically acceptable carrier.

Additional embodiments provide methods of decreasing OAS1 geneexpression or biological activity.

Additional embodiments provide for methods of specifically increasing ordecreasing the expression of certain forms of the OAS1 gene having atleast one mutation at position 2135728, 2135749, 2135978, 2144072,2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985,2156523, 2156595, or 2156638 of NT_(—)009775.13.

The invention provides an antisense oligonucleotide comprising at leastone modified internucleoside linkage.

The invention further provides an antisense oligonucleotide having aphosphorothioate linkage.

The invention still further provides an antisense oligonucleotidecomprising at least one modified sugar moiety.

The invention also provides an antisense oligonucleotide comprising atleast one modified sugar moiety which is a 2′-O-methyl sugar moiety.

The invention further provides an antisense oligonucleotide comprisingat least one modified nucleobase.

The invention still further provides an antisense oligonucleotide havinga modified nucleobase wherein the modified nucleobase is5-methylcytosine.

The invention also provides an antisense compound wherein the antisensecompound is a chimeric oligonucleotide.

The invention provides a method of inhibiting the expression of humanOAS1 in human cells or tissues comprising contacting the cells ortissues in vivo with an antisense compound or a ribozyme of 8 to 35nucleotides in length targeted to a nucleic acid molecule encoding humanOAS1 so that expression of human OAS1 is inhibited.

The invention further provides a method of decreasing or increasingexpression of specific forms of OAS1 in vivo, such forms being definedby having at least one mutation at position 2135728, 2135749, 2135978,2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294,2144985, 2156523, 2156595, or 2156638 of NT_(—)009775.13, usingantisense or RNAi compounds or ribozymes.

The invention further provides a method of modulating growth of cancercells comprising contacting the cancer cells in vivo with an antisensecompound or ribozyme of 8 to 35 nucleotides in length targeted to anucleic acid molecule encoding human OAS1 so that expression of humanOAS1 is inhibited.

The invention still further provides for identifying target regions ofOAS1 polynucleotides. The invention also provides labeled probes foridentifying OAS1 polynucleotides by in situ hybridization.

The invention provides for the use of an OAS1 inhibitor according to theinvention to prepare a medicament for preventing or inhibiting HCVinfection.

The invention further provides for directing an OAS1 inhibitor tospecific regions of the OAS1 protein or at specific functions of theprotein.

The invention also provides a pharmaceutical composition for inhibitingexpression of OAS1, comprising an antisense oligonucleotide according tothe invention in a mixture with a physiologically acceptable carrier ordiluent.

The invention further provides a ribozyme capable of specificallycleaving OAS1 RNA, and a pharmaceutical composition comprising theribozyme.

The invention also provides small molecule inhibitors of OAS1 whereinthe inhibitors are capable of reducing the activity of OAS1 or ofreducing or preventing the expression of OAS1 mRNA.

The invention further provides for inhibitors of OAS1 that modifyspecific functions of the protein other than the synthesis of 2′-5′oligoadenylates, such functions including interaction with otherproteins such as Hepatitis C virus NS5A protein.

The invention further provides for compounds that alterpost-translational modifications of OAS1 including but not limited toglycosylation and phosphorylation.

The invention further provides a human genetic screening method foridentifying an oligoadenylate synthetase gene mutation comprising: (a)treating, under amplification conditions, a sample of genomic DNA from ahuman with a polymerase chain reaction (PCR) primer pair for amplifyinga region of human genomic DNA containing nucleotide position 2135728,2135749, 2135978, 2144072, 2144088, 2144116, 2144321, 2131025, 2133961,2139587, 2144294, 2144985, 2156523, 2156595, or 2156638 ofoligoadenylate synthetase gene, said treating producing an amplificationproduct containing said region; and (b) detecting in the amplificationproduct of step (a) the presence of an nucleotide point mutation atnucleotide position 2135728, 2135749, 2135978, 2144072, 2144088,2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985, 2156523,2156595, or 2156638, thereby identifying said mutation.

In certain embodiments of this method, the region comprises a nucleotidesequence represented by a sequence selected from the group consisting ofSEQ ID NO:1-7 and SEQ ID NO:57-64. In other embodiments, the regionconsists essentially of a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1-7 and SEQ ID NO:57-64. Also provided is amethod of detecting, wherein the detecting comprises treating, underhybridization conditions, the amplification product of step (a) abovewith an oligonucleotide probe specific for the point mutation, anddetecting the formation of a hybridization product. In certainembodiments of the method, the oligonucleotide probe comprises anucleotide sequence selected from the group consisting of SEQ IDNO:12-18.

The invention also relates to a method for detecting in a human ahepatitis C infection resistance disease allele containing a pointmutation comprising substitution of a non wild-type nucleotide for awild-type nucleotide at nucleotide position 2135728, 2135749, 2135978,2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294,2144985, 2156523, 2156595, or 2156638 of oligoadenylate synthetase gene(OAS1), which method comprises: (a) forming a polymerase chain reaction(PCR) admixture by combining, in a PCR buffer, a sample of genomic DNAfrom said human and an oligoadenylate synthetase gene-specific PCRprimer pair selected from the group consisting of SEQ ID NO:8 and 9, andSEQ ID NO:10 and 11; (b) subjecting the PCR admixture to a plurality ofPCR thermocycles to produce an oligoadenylate synthetase geneamplification product; and (c) treating, under hybridization conditionsproducts produced in step (b), with a probe selected from the groupconsisting of SEQ ID NO:12-18, thereby detecting said mutation.

Also provided is an isolated OAS1 inhibitor selected from the groupconsisting of an antisense oligonucleotide, a ribozyme, a smallinhibitory RNA (RNAi), a protein, a polypeptide, an antibody, and asmall molecule. The isolated inhibitor may be an antisense molecule orthe complement thereof comprising at least 15 consecutive nucleic acidsof the sequence of SEQ ID NO:19. In other embodiments, the isolated OAS1inhibitor (antisense molecule or the complement thereof) hybridizesunder high stringency conditions to the sequence of SEQ ID NO:19.

The isolated OAS1 inhibitor may be selected from the group consisting ofan antibody and an antibody fragment. Also provided is a compositioncomprising a therapeutically effective amount of at least one OAS1inhibitor in a pharmaceutically acceptable carrier.

The invention also relates to a method of inhibiting the expression ofOAS1 in a mammalian cell, comprising administering to the cell an OAS1inhibitor selected from the group consisting of an antisenseoligonucleotide, a ribozyme, a protein, an RNAi, a polypeptide, anantibody, and a small molecule.

The invention further relates to a method of inhibiting the expressionof OAS1 gene expression in a subject, comprising administering to thesubject, in a pharmaceutically effective vehicle, an amount of anantisense oligonucleotide which is effective to specifically hybridizeto all or part of a selected target nucleic acid sequence derived fromsaid OAS1 gene.

The invention still further relates to a method of preventing infectionby a flavivirus in a human subject susceptible to the infection,comprising administering to the human subject an OAS1 inhibitor selectedfrom group consisting of an antisense oligonucleotide, a ribozyme, anRNAi, a protein, a polypeptide, an antibody, and a small molecule,wherein said OAS1 inhibitor prevents infection by said flavivirus.

The invention still further relates to a method of preventing or curinginfection by a flavivirus or other virus in a human subject susceptibleto the infection, comprising administering to the human subject an OAS1inhibitor selected from group consisting of an antisenseoligonucleotide, a ribozyme, an RNAI, a protein, a polypeptide, anantibody, and a small molecule, wherein said OAS1 inhibitor preventsinfection by said flavivirus or other virus and wherein said OAS1inhibitor is directed at one or more specific forms of the proteindefined by a mutation at position 2135728, 2135749, 2135978, 2144072,2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985,2156523, 2156595, or 2156638 of NT_(—)009775.13.

The invention still further relates to a method of preventing or curinginfection by a flavivirus or any other virus in a human subjectsusceptible to the infection by administering one of the polypeptides ofthe sequence: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:46, SEQ ID NO:47 and/or SEQ ID NO:48.

The invention embodies also treatments for infection with the humanimmunodeficiency virus (HIV).

The invention still further relates to a method of preventing insulindependent diabetes mellitus (IDDM) in a human subject, comprisingadministering to the human subject an OAS1 inhibitor selected from groupconsisting of an antisense oligonucleotide, a ribozyme, an RNAi, aprotein, a polypeptide, an antibody, and a small molecule, wherein saidOAS1 inhibitor prevents IDDM.

The invention still further relates to a method of preventing IDDM in ahuman subject, comprising administering to the human subject an OAS1inhibitor selected from group consisting of an antisenseoligonucleotide, a ribozyme, an RNAi, a protein, a polypeptide, anantibody, and a small molecule, wherein said OAS1 inhibitor preventsIDDM and wherein said OAS1 inhibitor is directed at one or more specificforms of the protein defined by a mutation at position 2135728, 2135749,2135978, 2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587,2144294, 2144985, 2156523, 2156595, or 2156638 of NT_(—)009775.13.

The invention still further relates to a method of treating cancer, suchas prostate cancer by increasing expression of the OAS1 gene or bytherapeutic administration of polypeptides disclosed herein.

Also provided is a method for inhibiting expression of a OAS1 targetgene in a cell in vitro comprising introduction of a ribonucleic acid(RNA) into the cell in an amount sufficient to inhibit expression of theOAS1 target gene, wherein the RNA is a double-stranded molecule with afirst strand consisting essentially of a ribonucleotide sequence whichcorresponds to a nucleotide sequence of the OAS1 target gene and asecond strand consisting essentially of a ribonucleotide sequence whichis complementary to the nucleotide sequence of the OAS1 target gene,wherein the first and the second ribonucleotide strands are separatecomplementary strands that hybridize to each other to form saiddouble-stranded molecule, and the double-stranded molecule inhibitsexpression of the target gene.

In certain embodiments of the method, the first ribonucleotide sequencecomprises at least 20 bases which correspond to the OAS1 target gene andthe second ribonucleotide sequence comprises at least 20 bases which arecomplementary to the nucleotide sequence of the OAS1 target gene. Instill further embodiments, the target gene expression is inhibited by atleast 10%.

In still further embodiments of the method, the double-strandedribonucleic acid structure is at least 20 bases in length and each ofthe ribonucleic acid strands is able to specifically hybridize to adeoxyribonucleic acid strand of the OAS1 target gene over the at least20 bases.

The invention provides a polypeptide or protein capable of restoringfunction of OAS1 that may be diminished or lost due to gene mutation. Insome embodiments the polypeptide or protein has the amino acid sequenceof wild type OAS1 encoded by a gene comprising SEQ ID NO:19. In otherembodiments, wherein a mutation in the OAS1 gene confers increasedactivity, stability, and/or half life on the OAS1 protein, or otherchange making the OAS1 protein more suitable for anti-viral activity,the protein or polypeptide encoded by the mutated OAS1 gene ispreferred.

Any of the foregoing proteins and polypeptides can be provided as acomponent of a therapeutic composition.

Also provided is the use of any of the proteins consisting of SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:46, SEQ IDNO:47 and/or SEQ ID NO:48 as a component of a therapeutic composition.

In a further embodiment, a nucleic acid encoding the OAS1 protein, OAS1mutant protein, or OAS1 polypeptide can be administered in the form ofgene therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a Table showing the locations of the mutations of the presentinvention in the OAS1 gene, the allelic variants (base substitutions),coordinates of the mutation on the genomic sequence, and NCBI dbSNP IDif any.

FIG. 2 (SEQ ID NO:19) is a polynucleotide sequence consisting of theconsecutive nucleotide bases at positions 2,130,000-2,157,999 of NCBIAccession No. NT_(—)009775.13, OAS1.

FIG. 3 shows SEQ ID NO: 20-64.

FIG. 4 shows approximate haplotype distributions within the OAS1 gene ina Caucasian population.

FIGS. 5A and B illustrates the variety of transcript variants disclosedby the invention and their relation to mutations disclosed by theinvention. FIG. 5A shows transcript Variant (TV) forms 1-6, and FIG. 5Bshows TV forms 7-10. FIG. 5C further illustrates the inferred structureof chimpanzee and gorilla variants relative to human.

FIG. 6 is a table describing non-human primate mutations in the OAS1gene relative to human and the coordinates of the mutations on thecorresponding human genomic sequence.

FIG. 7 demonstrates the enzymatic activity of exemplary polypeptides ofthe present invention when expressed and recovered from E. coli. Theactivity of oligoadenylate synthetase 1 enzymes produced in E. coli(Lanes 3-21) were compared to the enzyme produced in a baculovirusexpression system (Lane 2). E. coli samples were produced in either oneof two bacterial strains and diluted 1:10 (Lanes 3, 8, 13, and 18), 1:25(Lanes 4, 9, 14, and 19), 1:50, 1:100, and 1:200 as shown in the Figure.Enzyme reactions were carried out in the presence of α³²PdATP andenzymatic products were resolved by thin layer chromatography onpolyethylimine cellulose coated plates.

FIG. 8 demonstrates the enzymatic activity of exemplary polypeptidesresulting from polynucleotides of the present invention when expressedin HeLa cells. OAS1 isoforms were expressed in HeLa cells by transienttransfection, and crude cellular lysates were evaluated for enzymeactivity by their ability to catalyze the formation of oligoadenylatesusing α³²PdATP as a substrate. Oligoadenylates were separated bydenaturing gel electrophoresis in 12% acrylamide/bis-acrylamide gels.Crude HeLa extracts (H) were compared against enzyme expressed in, andpurified from, bacteria (B).

FIG. 9 demonstrates development of antibodies to exemplary polypeptidesof the present invention. Polyclonal antibodies were raised to syntheticpeptide sequences derived from the unique portion of each OAS1 form byrabbit immunization. Each antibody was tested against proteinsynthesized in vitro in a rabbit reticulocyte lysate coupledtranscription-translation system.

FIG. 10 is a table detailing exemplary protein transduction domainpolypeptides.

DETAILED DESCRIPTION OF THE INVENTION Introduction and Definitions

This invention relates to novel mutations in the oligoadenylatesynthetase gene, use of these mutations for diagnosis of susceptibilityor resistance to viral infection, to proteins encoded by a gene having amutation according to the invention, and to prevention or inhibition ofviral infection using the proteins, antibodies, and related nucleicacids. These mutations correlate with resistance of the carrier toinfection with flavivirus, particularly hepatitis C virus.

Much of current medical research is focused on identifying mutations anddefects that cause or contribute to disease. Such research is designedto lead to compounds and methods of treatment aimed at the diseasestate. Less attention has been paid to studying the genetic influencesthat allow people to remain healthy despite exposure to infectiousagents and other risk factors. The present invention represents asuccessful application of a process developed by the inventors by whichspecific populations of human subjects are ascertained and analyzed inorder to discover genetic variations or mutations that confer resistanceto disease. The identification of a sub-population segment that has anatural resistance to a particular disease or biological conditionfurther enables the identification of genes and proteins that aresuitable targets for pharmaceutical intervention, diagnostic evaluation,or prevention, such as prophylactic vaccination.

The sub-population segment identified herein is comprised of individualswho, despite repeated exposure to hepatitis C virus (HCV) havenonetheless remained sero-negative, while cohorts have become infected(sero-positive). The populations studied included hemophiliac patientssubjected to repeated blood transfusions, and intravenous drug users whobecome exposed through shared needles and other risk factors.

HCV infection involves a complex set of proteins and immune systemcomponents that work together to achieve a level of infection that,while it causes disease, can develop into low steady state of virus ininfected cells, apparently allowing HCV to escape from the hostimmuno-surveillance system, while enabling persistent viral infection.(Dansako et al., Virus Research 97:17-30, 2003.) The present inventionfocuses on one component of this system, the interferon-inducible2′-5′-oligoadenylate synthetase gene, specifically OAS1. The OAS1 geneplays a major role in the antiviral activity of host cells in the human,by activating ribonuclease L (RNase L) to cleave viral RNA. HCV RNAactivates the 2′-5′-OAS/RNase L pathway. As pointed out by Dansako etal., it may appear contradictory for HCV RNA to activate a pathway thatleads to cleavage of the viral RNA. However, such activity may serve toretain a balance between the host immune defense and a level ofinfection that would kill the host.

In view of this complex role of the OAS1 gene, it is of significantinterest that the present invention has identified a strong correlationbetween mutations in the OAS1 gene, and resistance to HCV infection incarriers of these mutations. The presence of such individuals nowpermits the elucidation of how OAS1 contributes to resistance to HCVinfection despite repeated exposure to infectious levels of the virus.This information will then lead to development of methods andcompositions for replicating the resistance mechanism in individualslacking natural resistance.

The present invention therefore provides that, regardless of themechanism, the mutations identified herein are useful for identifyingindividuals who are resistant to HCV infection. The resistance may comeabout through a loss of function of the OAS1 protein, in which case itis predicted that HCV viral levels would be high enough to prevent thevirus from escaping from the host immuno-surveillance system, hencefacilitating destruction of the virus. The resistance may also comeabout through gain of function in that the OAS1 protein level isenhanced, the half life of the protein is increased, and/or the proteinstructure is affected in a way that enhances its ability to activateribonuclease L to cleave viral RNA. The resistance may also come aboutthrough modifications to the OAS1 protein that prevent inhibition ofnormal OAS1 protein function by HCV viral proteins or nucleotides. Theresistance may also come about through modifications to the OAS1 proteinthat prevent interaction of the protein with HCV viral proteins ornucleotides that are necessary for the normal HCV viral lifespan. Theinvention is not limited to one mechanism. Furthermore, although severaldifferent point mutations are disclosed herein, this is not intended tobe indicative that each mutation has the same effect on OAS1 proteinstructure or function.

OAS1 plays a role in infection by other viruses of the flavivirusfamily, of which HCV is a member. The flavivirus family also includesviruses that cause yellow fever, dengue fever, St. Louis encephalitis,Japanese encephalitis, and other viral diseases disclosed herein. Thehost defense to these viruses includes virus-inducible interferon. Theinterferon induces 2′-5-oligoadenylate synthetases, which as discussedabove, are involved in the activation of RNaseL. RNaseL in turn cleavesviral RNA. Other viral infections may by amenable to prevention and/orinhibition by the methods disclosed herein, including RSV.

In reference to the detailed description and preferred embodiment, thefollowing definitions are used:

A: adenine; C: cytosine; G: guanine; T: thymine (in DNA); and U: uracil(in RNA)

Allele: A variant of DNA sequence of a specific gene. In diploid cells amaximum of two alleles will be present, each in the same relativeposition or locus on homologous chromosomes of the chromosome set. Whenalleles at any one locus are identical the individual is said to behomozygous for that locus, and when they differ the individual is saidto be heterozygous for that locus. Since different alleles of any onegene may vary by only a single base, the possible number of alleles forany one gene is very large. When alleles differ, one is often dominantto the other, which is said to be recessive. Dominance is a property ofthe phenotype and does not imply inactivation of the recessive allele bythe dominant. In numerous examples the normally functioning (wild-type)allele is dominant to all mutant alleles of more or less defectivefunction. In such cases the general explanation is that one functionalallele out of two is sufficient to produce enough active gene product tosupport normal development of the organism (i.e., there is normally atwo-fold safety margin in quantity of gene product).

Haplotype: One of many possible pluralities of Alleles, serially orderedby chromosomal localization and representing that set of Alleles carriedby one particular homologous chromosome of the chromosome set.

Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1′ carbon of thepentose) and that combination of base and sugar is a nucleoside. Whenthe nucleoside contains a phosphate group bonded to the 3′ or 5′position of the pentose it is referred to as a nucleotide. A sequence ofoperatively linked nucleotides is typically referred to herein as a“base sequence” or “nucleotide sequence”, and their grammaticalequivalents, and is represented herein by a formula whose left to rightorientation is in the conventional direction of 5′-terminus to3′-terminus.

Base Pair (bp): A partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA,uracil (U) is substituted for thymine. When referring to RNA herein, thesymbol T may be used interchangeably with U to represent uracil at aparticular position in the RNA molecule.

Nucleic Acid: A polymer of nucleotides, either single or doublestranded.

Polynucleotide: A polymer of single or double stranded nucleotides. Asused herein “polynucleotide” and its grammatical equivalents willinclude the full range of nucleic acids. A polynucleotide will typicallyrefer to a nucleic acid molecule comprised of a linear strand of two ormore deoxyribonucleotides and/or ribonucleotides. The exact size willdepend on many factors, which in turn depends on the ultimate conditionsof use, as is well known in the art. The polynucleotides of the presentinvention include primers, probes, RNA/DNA segments, oligonucleotides or“oligos” (relatively short polynucleotides), genes, vectors, plasmids,and the like.

RNAi: RNA interference (RNAi) is a method whereby small interfering RNA(siRNA), a duplex typically about 21-23 nucleotides long, is introducedinto a cell, leading ultimately to the degradation of messenger RNA of atargeted gene containing an identical or complementary sequence andeffectively silencing it.

Gene: A nucleic acid whose nucleotide sequence codes for an RNA orpolypeptide. A gene can be either RNA or DNA.

Duplex DNA: A double-stranded nucleic acid molecule comprising twostrands of substantially complementary polynucleotides held together byone or more hydrogen bonds between each of the complementary basespresent in a base pair of the duplex. Because the nucleotides that forma base pair can be either a ribonucleotide base or a deoxyribonucleotidebase, the phrase “duplex DNA” refers to either a DNA-DNA duplexcomprising two DNA strands (ds DNA), or an RNA-DNA duplex comprising oneDNA and one RNA strand.

Complementary Bases: Nucleotides that normally pair up when DNA or RNAadopts a double stranded configuration.

Complementary Nucleotide Sequence: A sequence of nucleotides in asingle-stranded molecule of DNA or RNA that is sufficientlycomplementary to that on another single strand to specifically hybridizeto it with consequent hydrogen bonding.

Conserved: A nucleotide sequence is conserved with respect to apreselected (reference) sequence if it non-randomly hybridizes to anexact complement of the preselected sequence.

Hybridization: The pairing of substantially complementary nucleotidesequences (strands of nucleic acid) to form a duplex or heteroduplex bythe establishment of hydrogen bonds between complementary base pairs. Itis a specific, i.e. non-random, interaction between two complementarypolynucleotides that can be competitively inhibited.

Nucleotide Analog: A purine or pyrimidine nucleotide that differsstructurally from A, T, G, C, or U, but is sufficiently similar tosubstitute for the normal nucleotide in a nucleic acid molecule.

DNA Homolog: A nucleic acid having a preselected conserved nucleotidesequence and a sequence coding for a receptor capable of binding apreselected ligand.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3′- to 5′-directionalong the non-coding strand of the DNA or 5′-to 3′-direction along theRNA transcript.

Stop Codon: Any of three codons that do not code for an amino acid, butinstead cause termination of protein synthesis. They are UAG, UAA andUGA and are also referred to as a nonsense or termination codon.

Reading Frame: Particular sequence of contiguous nucleotide triplets(codons) employed in translation. The reading frame depends on thelocation of the translation initiation codon.

Intron: Also referred to as an intervening sequence, a noncodingsequence of DNA that is initially copied into RNA but is cut out of thefinal RNA transcript.

Modes of Practicing the Invention

The present invention provides a novel method for screening humans foroligoadenylate synthetase alleles associated with resistance toinfection by a flavivirus, particularly hepatitis C. The invention isbased on the discovery that such resistance is associated with a pointmutation (base substitution) in the oligoadenylate synthetase gene DNAsequence at nucleotide position 2135728, 2135749, 2135978, 2144072,2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985,2156523, 2156595, or 2156638 of Genbank Accession No. NT_(—)009775.13(SEQ ID NO:19), which encodes the human OAS1 gene.

This invention discloses the results of a study that identifiedpopulations of subjects resistant or partially resistant to infectionwith the hepatitis C virus (HCV) and that further identified geneticmutations that confer this beneficial effect. Several genetic mutationsin the 2′-5′-oligoadenylate synthetase genes are identified, that aresignificantly associated with resistance to HCV infection. The studydesign used was a case-control, allele association analysis. Casesassigned as subjects had serially documented or presumed exposure toHCV, but who did not develop infection as documented by the developmentof antibodies to the virus (i.e. HCV seronegative). Control subjectswere serially exposed subjects who did seroconvert to HCV positive. Caseand control subjects were recruited from three populations, hemophiliapatients from Vancouver, British Columbia, Canada; hemophilia patientsfrom Northwestern France; and injecting drug users from the Seattlemetropolitan region.

Case and control definitions differed between the hemophilia and IDUgroups and were based upon epidemiological models of infection riskpublished in the literature and other models developed by the inventors,as described herein. For the hemophilia population, control subjectswere documented to be seropositive for antibodies to HCV usingcommercial diagnostics laboratory testing. Case subjects were documentedas being HCV seronegative, having less than 5% of normal clottingfactor, and having received concentrated clotting factors before January1987. Control injecting drug users were defined as documented HCVseropositive. Case injecting drug users were defined as documented HCVseronegative, having injected drugs for more than ten years, and havingreported engaging in one or more additional risk behaviors. Additionalrisk behaviors include the sharing of syringes, cookers, or cottons withanother IDU. In one particular Caucasian population 20 cases and 42controls were included in the study.

Selection of case and control subjects was performed essentially asdescribed in U.S. patent application Ser. No. 09/707,576 using thepopulation groups at-risk affected (“controls”) and at-risk unaffected(“cases”).

The present inventive approach to identifying gene mutations associatedwith resistance to HCV infection involved the selection of candidategenes. Approximately 50 candidate genes involved in viral binding to thecell surface, viral propagation within the cell, the interferonresponse, and aspects of the innate immune system and the antiviralresponse, were interrogated. Candidate genes were sequenced in cases andcontrols by using the polymerase chain reaction to amplify targetsequences from the genomic DNA of each subject. PCR products fromcandidate genes were sequenced directly using automated,fluorescence-based DNA sequencing and an ABI3730 automated sequencer.

Genetic mutations were identified in an oligoadenylate synthetase gene(OAS1) that either alone, or in combination, were significantlyassociated (p<0.05) with resistance to HCV infection. The basesubstitutions and deletions that constitute these mutations are shown inFIG. 1. Variant forms of the OAS1 gene (“OAS1R”) are produced by thepresence of one or more of the mutations of the present invention(identified as SEQ ID NO:1-7 and SEQ ID NO:57-64). These variant OAS1Rforms of the OAS1 gene are believed to confer resistance to viralinfection.

Population and subject parental haplotypes, comprising pluralities ofthe OAS1 mutations (SEQ ID NO:1-7 and SEQ ID NO:57-64), arecomputationally inferred from the case-control genotyping data set byExpectation Maximation methods as known to those skilled in the art(Excoffier and Slatkin, Mol. Biol. Evol. 12:921-927, 1995). Theinvention embraces both the use of the full range of OAS1 mutations forhaplotypic analysis as well as the use of subsets of OAS1 mutations forcomputational convenience. Comparative analysis of patterns ofsegregation of haplotypes and haplotype subsets with the case andcontrol groups identify mutations of particular potency with regard toviral resistance or susceptibility.

In one illustrative example, haplotypes are computed comprisingpluralities of the OAS1 mutations identified by SEQ ID NO: 2 through SEQID NO: 6. Several haplotypes are identified in a Caucasian case andcontrol population by this analysis. The definition of these haplotypesis shown in FIG. 4. Two common haplotypes (identified as HAP1 and HAP2)are identified that account for approximately 85% of inferred haplotypesand are in Hardy-Weinberg equilibrium, particularly with regard to theoccurrence of haplotype homozygotes in the population. Further analysisof OAS1 in various human populations and in primates indicates that HAP2is the ancestral primate haplotype pre-dating the divergence of oldworld monkeys and hominids. One additional haplotype (identified asHAP3) is associated with the persistently HCV-resistant case group inthis particular population. Therefore subjects carrying the HAP3haplotype are at substantially lower risk of HCV infection. The HAP3haplotype appears to have arisen through a complex series ofrecombination and mutation originating from the ancestral haplotype. Thecombined rarity of such events combined with the considerable occurrenceof haplotype HAP3 suggests positive selection acted to develop andretain haplotype HAP3 in the population, possibly as a response torecurring viral challenge over time. In this example, haplotype HAP3 isthe only haplotype occurring at an appreciable population frequency thatcombines the effects of a G nucleotide for mutation SEQ ID NO:2 togetherwith an A nucleotide for mutation SEQ ID NO:4 in a single pre-cursorRNA.

The present invention is not limited by the foregoing illustrativeexample. Nor is the present invention limited by other illustrativeexamples that provide insight into the relevance and utility ofparticular OAS1 mutations. In another illustrative example, thesubstitution of a G nucleotide for an A nucleotide in SEQ ID NO:2results in a predicted amino acid substitution of a Serine to Glycine.Computational prediction as known to those skilled in the art is highlysuggestive that the Serine is a site of phosphorylation whereas theGlycine would not be phosphorylated.

In a further illustrative example, the substitution of an A nucleotidefor a G nucleotide in mutation SEQ ID NO:4 occurs in the consensussplice acceptor site for the wild-type sixth exon in OAS1. Thissubstitution replaces the requisite G in the splice acceptor recognitionsignal but in the process creates a new splice recognition site one basepair downstream. The mutated form thereby creates a frameshift in thetranslated protein. The mutated site also is a less effective splicesignal and as a result encourages additional alternative splicing ofpre-cursor RNAs in addition to the frameshifted exon 6 splicing.Preferred embodiments of these alternative splice forms are provided inFIGS. 5A and 5B. Further illustrative examples of genetic analysis areprovided below.

Promiscuous splicing of OAS1 transcript variants was independentlyconfirmed by reverse transcription of RNA derived from both lymphocytecell lines and peripheral blood mononuclear cells (PBMCs) isolated fromfresh human serum. PCR analysis of reverse transcribed RNA products fromvarious haplotype carrying cell lines and PBMCs indicated RNA formscarrying A nucleotides for mutation SEQ ID NO:4 resulted in a multitudeof transcript variant OAS1R forms. These OAS1R transcript variants aredepicted graphically in FIGS. 5A and 5B and comprise SEQ ID NO:36through SEQ ID NO:47 as provided in FIG. 3.

These variant OAS1R forms of the OAS1 gene and corresponding transcriptvariants are believed to encode one or more of the polypeptidesconsisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO: 35, SEQ ID NO: 46, and/orSEQ ID NO: 47. The foregoing polypeptides, either singly or plurally,may be referred to herein as OAS1R polypeptides or OAS1R proteinsinterchangeably. A common feature of many of the foregoing polypeptidesis that they differ primarily in their carboxyl-terminus whileconserving the amino-terminal portion.

In addition to the production of alternative transcripts themselves, theOAS1R forms of the gene may also contain or abolish specific sequencecontexts (such as Exon Splice Enhancers) that modify the selectivepreference for specific transcript variants. This in turn would causediffering relative levels of abundance of the resulting proteins. Thesevariant OAS1R forms of the OAS1 gene may also modify localization orpost-translational modification of the resulting proteins. Those skilledin the art will appreciate that increased abundance or othermodifications that improve the activity, stability, or availability of aspecific OAS1 protein form may improve the overall anti-viralperformance of the 2′-5′-OAS/RNase L pathway. Those skilled in the artcan likewise appreciate that depressing the activity or availability ofa specific OAS1 form may also improve the overall anti-viral performanceof the 2′-5′-OAS/RNase L pathway in cases where said specific protein isnot advantaged, or even disadvantaged, over other specific OAS1 forms.Without limitation, one embodiment of a disadvantaged OAS1 protein isone which is specifically targeted by viral protein(s) in such a manneras to preclude the enzymatic activity of said specific OAS1 protein. Afurther embodiment of a non-advantaged OAS1 protein is one with lowerenzymatic activity polymerizing with other active forms therebylowering, or abolishing, the overall enzymatic activity (and hencedecreasing overall anti-viral effect) of the polymerized protein. One ormore of the foregoing mechanisms may contribute to resistance to viralinfection. The present invention is not limited, however, by thespecific mechanism of action of the disclosed variant polynucleotides orpolypeptides.

The invention therefore provides novel forms of the human2′-5′-oligoadenylate synthetase gene, novel mRNA transcripts, andassociated proteins. The invention also discloses utility for the novelmRNA transcripts and novel proteins. These novel forms are characterizedby the presence in the gene of one or more of several rare geneticmutations or haplotypes not disclosed in the public databases. Thesenovel forms of OAS1, OAS1R, confer on carriers a level of resistance tothe hepatitis C virus and associated flaviviruses including but notlimited to the West Nile virus, dengue viruses, yellow fever virus,tick-borne encephalitis virus, Japanese encephalitis virus, St. Louisencephalitis virus, Murray Valley virus, Powassan virus, Rocio virus,louping-ill virus, Banzi virus, Ilheus virus, Kokobera virus, Kunjinvirus, Alfuy virus, bovine diarrhea virus, and the Kyasanur forestdisease virus. The OAS proteins have also been shown to be important inattenuating infection in experimental respiratory syncitial virus andpicornavirus cell culture infection systems. Failure of humanimmunodeficiency virus-1 (HIV-1) infected cells to release virus hasbeen correlated with high concentrations of OAS and/or 2-5A.Furthermore, HIV-1 transactivator protein (tat) has been shown to blockactivation of OAS (Muller et al, J Biol Chem. 1990 Mar. 5;265(7):3803-8) thus indicating that novel forms of OAS might evade HIV-1defense mechanisms and provide an effective therapy. Thus, these OAS1Rforms of OAS1 disclosed herein may confer resistance to thesenon-flavivirus infectious agents as well.

The present invention also provides novel description of the chimpanzee(Pan troglodytes) and gorilla (Gorilla gorilla) forms of OAS1, each ofwhich leads to a novel mRNA and polypeptide with utility. While genesare typically very highly conserved in closely related primates, such ashumans, chimpanzees and gorillas, important differences in OAS1 areobserved between the three species. Chimpanzees, the closest humanrelative, possess a single base substitution within OAS1 exon 5 (at asite equivalent to 2,142,351 in NT_(—)009775.13 in humans and defined bySEQ ID NO: 53) that causes a truncated protein product. The chimpanzeeOAS1 polypeptide and mRNA sequences are provided by SEQ ID NO: 51 andSEQ ID NO: 55, respectively. Gorillas also possess a two base pairdeletion (at the sites equivalent to 2,144,089-2,144,090 inNT_(—)009775.13 in humans and defined by SEQ ID NO: 54) within exon 6near the acceptor splice site that causes a premature stop oftranslation. The gorilla partial polypeptide and partial mRNA sequencesare provided by SEQ ID NO: 52 and SEQ ID NO: 56, respectively. Theinferred structure of the chimpanzee and gorilla transcripts relative tohuman is provided in FIG. 5C. Each of these cases, like the human OAS1Rpolypeptides, possess highly conserved polypeptide sequences that differmost notably in the structure and content of the carboxyl-terminal tail.The common amino-terminal portion of these polypeptides contains all ofthe elements previously demonstrated to be required for OAS1 enzymaticactivity. As chimpanzees and gorillas have been subjected to viralchallenges similar to that of humans in Africa, the prevalence of thesedistinct but functionally similar primate variants provides furtherevidence that the carboxy-terminal portions of longer OAS1 forms areunnecessary or even disadvantaging in surviving viral challenges. Thefact that chimpanzees possess an outright truncation of the OAS1polypeptide as opposed to the heterogeneity of transcript variants inhumans is consistent with the observation that chimpanzees, while theonly other primate known to be susceptible to HCV infection, do have anatypical infection relative to humans characterized by an increasedfrequency of viral clearance and the absence of resulting fibrosis orhepatocellular carcinoma.

Each novel OAS1R cDNA is cloned from human subjects who are carriers ofthese mutations. Cloning is carried out by standard cDNA cloning methodsthat involve the isolation of RNA from cells or tissue, the conversionof RNA to cDNA, and the conversion of cDNA to double-stranded DNAsuitable for cloning. As one skilled in the art will recognize, all ofthese steps are routine molecular biological analyses. Other methodsinclude the use of reverse transcriptase PCR, 5′RACE (RapidAmplification of cDNA Ends), or traditional cDNA library constructionand screening by Southern hybridization. All OAS1R alleles describedherein are recovered from patient carriers. Each newly cloned OAS1R cDNAis sequenced to confirm its identity and to identify any additionalsequence differences relative to wild-type.

Novel OAS1R gene mutations may affect resistance to viral infection bymodifying the properties of the resulting OAS1 mRNA. Therefore,differences in mRNA stability between carriers of the OAS1R alleles andhomozygous wild-type subjects are evaluated. RNA stability is evaluatedand compared using known assays including Taqman® and simple Northernhybridization. These constitute routine methods in molecular biology.

OAS1R mutations may affect infection resistance by modifying theregulation of the OAS1 gene. It is known that expression of OAS genes isinduced by interferon treatment and during viral infection. The OAS1Ralleles may confer resistance to viral infection through constitutiveexpression, over-expression, or other disregulated expression. Severalmethods are used to evaluate gene expression with and without interferonor viral stimulation. These methods include expression microarrayanalysis, Northern hybridization, Taqman®, and others. Samples arecollected from tissues known to express the OAS genes such as theperipheral blood mononuclear cells. Gene expression is compared betweentissues from OAS1R carriers and non-carriers. In one embodiment,peripheral blood mononuclear cells are collected from carriers and noncarriers, propagated in culture, and stimulated with interferon. Thelevel of expression of OAS1R alleles during interferon induction iscompared to wild-type alleles. In another embodiment, human subjects aretreated with interferon and the level of induction of the OAS1 gene isevaluated in carriers of the OAS1R mutations versus non-carriers. As oneskilled in the art can appreciate, numerous combinations of tissues,experimental designs, and methods of analysis are used to evaluate OAS1Rgene regulation.

Once the novel cDNA for each OAS1R is cloned, it is used to manufacturerecombinant OAS1R proteins using any of a number of different knownexpression cloning systems. In one embodiment of this approach, an OAS1RcDNA is cloned by standard molecular biological methods into anEscherichia coli expression vector adjacent to an epitope tag thatcontains a sequence of DNA coding for a polyhistidine polypeptide. Therecombinant protein is then purified from Escherichia coli lysates usingimmobilized metal affinity chromatography or similar method. One skilledin the art will recognize that there are many different expressionvectors and host cells that can be used to purify recombinant proteins,including but not limited to yeast expression systems, baculovirusexpression systems, Chinese hamster ovary cells, and others.

Computational methods are used to identify short peptide sequences fromOAS1R proteins that uniquely distinguish these proteins from wild-typeOAS1 proteins. Various computational methods and commercially availablesoftware packages can be used for peptide selection. Thesecomputationally selected peptide sequences can be manufactured using theFMOC peptide synthesis chemistry or similar method. One skilled in theart will recognize that there are numerous chemical methods forsynthesizing short polypeptides according to a supplied sequence.

Peptide fragments and the recombinant protein from the OAS1R gene can beused to develop antibodies specific to this gene product. As one skilledin the art will recognize, there are numerous methods for antibodydevelopment involving the use of multiple different host organisms,adjuvants, etc. In one classic embodiment, a small amount (150micrograms) of purified recombinant protein is injected subcutaneouslyinto the backs of New Zealand White Rabbits with subsequent similarquantities injected every several months as boosters. Rabbit serum isthen collected by venipuncture and the serum, purified IgG, or affinitypurified antibody specific to the immunizing protein can be collected.As one skilled in the art will recognize, similar methods can be used todevelop antibodies in rat, mouse, goat, and other organisms. Peptidefragments as described above can also be used to develop antibodiesspecific to the OAS1R protein. The development of both monoclonal andpolyclonal antibodies is suitable for practicing the invention. Thegeneration of mouse hybridoma cell lines secreting specific monoclonalantibodies to the OAS1R proteins can be carried out by standardmolecular techniques.

Antibodies prepared as described above can be used to develop diagnosticmethods for evaluating the presence or absence of the OAS1R proteins incells, tissues, and organisms. In one embodiment of this approach,enzyme-linked immunosorbent assays can be developed using purifiedrecombinant OAS1R proteins and specific antibodies in order to detectthese proteins in human serum. These diagnostic methods can be used tovalidate the presence or absence of OAS1R proteins in the tissues ofcarriers and non-carriers of the above-described genetic mutations.

Antibodies prepared as described above can also be used to purify nativeOAS1R proteins from those patients who carry these mutations. Numerousmethods are available for using antibodies to purify native proteinsfrom human cells and tissues. In one embodiment, antibodies can be usedin immunoprecipitation experiments involving homogenized human tissuesand antibody capture using protein A. This method enables theconcentration and further evaluation of mutant OAS1R proteins. Numerousother methods for isolating the native forms of OAS1R are availableincluding column chromatography, affinity chromatography, high pressureliquid chromatography, salting-out, dialysis, electrophoresis,isoelectric focusing, differential centrifugation, and others.

Proteomic methods are used to evaluate the effect of OAS1R mutations onsecondary, tertiary, and quaternary protein structure. Proteomic methodsare also used to evaluate the impact of OAS1R mutations on thepost-translational modification of the OAS protein. There are many knownpossible post-translational modifications to a protein includingprotease cleavage, glycosylation, phosphorylation, sulfation, theaddition of chemical groups or complex molecules, and the like. A commonmethod for evaluating secondary and tertiary protein structure isnuclear magnetic resonance (NMR) spectroscopy. NMR is used to probedifferences in secondary and tertiary structure between wild-type OAS1proteins and OAS1R proteins. Modifications to traditional NMR are alsosuitable, including methods for evaluating the activity of functionalsites including Transfer Nuclear Overhauser Spectroscopy (TrNOESY) andothers. As one skilled in the art will recognize, numerous minormodifications to this approach and methods for data interpretation ofresults can be employed. All of these methods are intended to beincluded in practicing this invention. Other methods for determiningprotein structure by crystallization and X-ray diffraction are employed.

Mass spectroscopy can also be used to evaluate differences betweenmutant and wild-type OAS proteins. This method can be used to evaluatestructural differences as well as differences in the post-translationalmodifications of proteins. In one typical embodiment of this approach,the wild-type OAS1 protein and mutant OAS1R proteins are purified fromhuman peripheral blood mononuclear cells using one of the methodsdescribed above. These cells can be stimulated with interferon, asdescribed above, in order to increase expression of the OAS proteins.Purified proteins are digested with specific proteases (e.g. trypsin)and evaluated using mass spectrometry. As one skilled in the art willrecognize, many alternative methods can also be used. This inventioncontemplates these additional alternative methods. For instance, eithermatrix-assisted laser desorption/ionization (MALDI) or electrosprayionization (ESI) mass spectrometric methods can be used. Furthermore,mass spectroscopy can be coupled with the use of two-dimensional gelelectrophoretic separation of cellular proteins as an alternative tocomprehensive pre-purification. Mass spectrometry can also be coupledwith the use of peptide fingerprint database and various searchingalgorithms. Differences in post-translational modification, such asphosphorylation or glycosylation, can also be probed by coupling massspectrometry with the use of various pretreatments such as withglycosylases and phosphatases. All of these methods are to be consideredas part of this application.

OAS1 is active as a tetramer, and mutations that interfere withself-association affect enzyme activity. Known methods are used toevaluate the effect of OAS1R mutations on tetramer formation. Forinstance, immunoprecipitation with OAS1 and OAS1R specific antibodies isperformed in order to isolate OAS1/OAS1R complexes from patient cells,cell culture, or transfected cells over-expressing the OAS1 or OAS1R.These complexes can then be evaluated by gel electrophoresis or otherchromatographic methods which are well known to those skilled in theart.

OAS1 may confer viral resistance by interaction with other proteins. Theenzyme contains a region with structural homology to the BH3 domain ofthe bcl-2 family. This domain may be critical to the function of OAS1.According to the invention, OAS1-specific antibodies can be used toisolate protein complexes involving the OAS1 proteins from a variety ofsources as discussed above. These complexes can then be evaluated by gelelectrophoresis to separate members of the interacting complex. Gels canbe probed using numerous methods including Western blotting, and novelinteracting proteins can be isolated and identified using peptidesequencing. Differences in the content of OAS complexes in wild-type andOAS1R extracts will also be evaluated. As one skilled in the art willrecognize, the described methods are only a few of numerous differentapproaches that can be used to purify, identify, and evaluateinteracting proteins in the OAS complex. Additional methods include, butare not limited to, phage display and the use of yeast two-hybridmethods.

OAS1 is known to interact with hepatitis C virus NS5A protein (Taguchi,T. et al., J. Gen. Virol. 85:959-969, 2004). Without being bound by amechanism, the invention therefore relates to OAS1 proteins that do notinteract with the NS5A protein, wherein the proteins are polypeptides ofthe present invention, expressed by polynucleotides of the presentinvention, expressed by mRNA encoded by splice variants of OAS1, by OAS1polynucleotides containing at least one mutation of the presentinvention, by OAS1 polynucleotides having at least one mutation in thecoding region, and or by OAS1 polynucleotides having at least one basesubstitution, deletion or addition wherein binding to NS5A protein isaltered or prevented.

NS5A protein may exert a biological activity by inhibiting the antiviralactivity of interferon. This antiviral activity is in one model normallyimplemented when interferon stimulates OAS1 activity in the presence ofa co-factor, such as double stranded RNA. OAS1 polymerizes ATP into2′-5′-linked oligoadenylates, which activate RNase L to cleave singlestranded RNA including mRNA. Binding of NS5A to OAS1 can inhibit itsactivity, thereby inhibiting or preventing the cascade of activitiesthat would otherwise lead to destruction of the viral RNA.

Although the invention is not dependent on this model, the binding ofNS5A to OAS1 is consistent with a model in which mutated forms of OAS1avoid NS5A binding and inhibition and are thereby able to carry out thenormal function of polymerizing ATP. In such cases, consistent with theclinical results described herein, a person carrying such a mutation isresistant to infection by hepatitis C virus. Similarly, the truncatedform of OAS1 possessed by chimpanzees, as disclosed above, may eludebinding by NS5A or other viral proteins and thereby allow the observedhigher frequency of chimpanzee viral clearance. The mutation may in somecases directly affect the binding site of OAS1 for NS5A. In other casesthe mutation may be at a site separate from the actual binding site, butcauses a conformational change such that binding of OAS1 to NS5A isinhibited, slowed, or prevented.

The binding of OAS1 to NS5A in a physiologically and pathologicallyrelevant manner therefore provides an objective test for assaying theeffect of a base mutation, deletion or addition in an OAS1polynucleotide on a biological function of the encoded OAS1 protein.Such binding is assayed in a manner known in the art. In one exemplarybut not limiting method, such as described by Taguchi, T. et al. (J.Gen. Virol. 85:959-969, 2004), HeLa cells are transiently transfectedwith expression plasmids encoding GST-tagged NS5A and HA-tagged OAS1. ByOAS1 in this example is meant OAS1 according to the invention, includingOAS1 encoded by splice variants of OAS1, by OAS1 polynucleotides havingat least one mutation in the coding region, and/or by OAS1polynucleotides having at least one base substitution, deletion oraddition. After an appropriate incubation time such as 12-16 hours, thecells are washed, lysed, and centrifuged, and the resulting supernatantis mixed with glutathione-conjugated Sepharose beads, which aid inseparating GST-tagged proteins. Complexes of GST-tagged NS5A andHA-tagged OAS1 protein are identified by using and imaging antibodies toNS5A and anti-HA antibody. Variations on this method include using othertags for the individual proteins, such as FLAG-tag. In the context ofthe present invention, the main variable is the OAS1 protein orpolypeptide. The ability of the OAS1 protein or polypeptide to carry outone biologically relevant activity (i.e. the binding to a hepatitis Cprotein that is known to be protective for the ability of the virus toreplicate in the host) is objectively tested using these assays. OAS1proteins and polypeptides that do not bind to NS5A are suitablecandidates as therapeutic proteins.

The OAS1 proteins are enzymes that catalyze the conversion of ATP intooligoadenylate molecules. Several methods are available to evaluate theactivity of OAS1 enzymes. These methods are employed to determine theeffects of OAS1R mutations on the activity of the mutant proteinsrelative to the wild type enzyme. For example, oligoadenylate synthesisactivity can be measured by quantifying the incorporation of³²P-radiolabeled ATP into polyadenylates. The radiolabeledpolyadenylates can be quantified and characterized in terms of length bya number of chromatographic methods including electrophoresis or ionexchange chromatography. These assays also enable characterization ofsubstrate (ATP) binding and enzyme kinetics. OAS1 is activated by dsRNA.The kinetics of this activation is analyzed in OAS1 and compared toOAS1R using the activity assays described herein and synthetic dsRNAs asdescribed in the art.

The polypeptides of the present invention are demonstrated by these andother methods known in the art to possess oligoadenylate synthesisactivity. FIGS. 7 and 8 demonstrate the activities of several exemplarypolypeptides of the present invention. Regardless of their quantitativelevel of activity, this capacity to produce 2′-5′-oligodenylates is wellunderstood by those skilled in the art to produce anti-viral effectsthrough the activation of RNaseL. As such, the mere fact that thepolypeptides of the present invention possess oligoadenylate synthesisactivity indicates that said polypeptides have utility, particularly inconsideration of therapeutic uses thereof which are disclosed below.

Biological studies are performed to evaluate the degree to which OAS1Rmutant genes protect from viral infection. These biological studiesgenerally take the form of introducing the mutant OAS1R genes orproteins into cells or whole organisms, and evaluating their biologicaland antiviral activities relative to wild-type controls. In one typicalembodiment of this approach, the OAS1R genes are introduced into AfricanGreen monkey kidney (Vero) cells in culture by cloning the cDNAsisolated as described herein into a mammalian expression vector thatdrives expression of the cloned cDNA from an SV40 promoter sequence.This vector will also contain SV40 and cytomegalovirus enhancer elementsthat permit efficient expression of the OAS1R genes, and a neomycinresistance gene for selection in culture. The biological effects ofOAS1R expression can then be evaluated in Vero cells infected with thedengue virus. In the event that OAS1R confers broad resistance tomultiple flaviviruses, one would expect an attenuation of viralpropagation in cell lines expressing these mutant forms of OAS1 relativeto wild-type. As one skilled in the art will recognize, there aremultiple different experimental approaches that can be used to evaluatethe biological effects of OAS1R genes and proteins in cells andorganisms and in response to different infectious agents. For instance,in the above example, different expression vectors, cell types, andviral species may be used to evaluate the OAS1R resistance effects.Primary human cells in culture may be evaluated as opposed to celllines. Cells may be stimulated with double-stranded RNA or interferonbefore introduction of the virus. Expression vectors containingalternative promoter and enhancer sequences may be evaluated. Virusesother than the flaviviruses (e.g. respiratory syncytial virus andpicornavirus) are evaluated.

Transgenic animal models are developed to assess the usefulness ofmutant forms of OAS1 in protecting against whole-organism viralinfection. In one embodiment, OAS1R genes are introduced into thegenomes of mice susceptible to flavivirus infection (e.g. the C3H/Heinbred laboratory strain). These OAS1R genes are evaluated for theirability to modify infection or confer resistance to infection insusceptible mice. As one skilled in the art will appreciate, numerousstandard methods can be used to introduce transgenic human OAS1R genesinto mice. These methods can be combined with other methods that affecttissue specific expression patterns or that permit regulation of thetransgene through the introduction of endogenous chemicals, the use ofinducible or tissue specific promoters, etc.

As a model for hepatitis C infection, cell lines expressing OAS1R genescan be evaluated for susceptibility, resistance, or modification ofinfection with the bovine diarrheal virus (BVDV). BVDV is a commonlyused model for testing the efficacy of potential anti-HCV antiviraldrugs (Buckwold et. al., Antiviral Research 60:1-15, 2003). In oneembodiment, the OAS1R genes can be introduced into KL (calf lung) cellsusing expression vectors essentially as described above and tested fortheir ability to modify BVDV infection in this cell line. Furthermore,mouse models of HCV infection (e.g. the transplantation of human liversinto mice, the infusion of human hepatocyte into mouse liver, etc.) mayalso be evaluated for modification of HCV infection in the transgenicsetting of OAS1R genes. Experiments can be performed whereby the effectsof expression of OAS1R genes are assessed in HCV viral culture systems.

Cell culture systems can also be used to assess the impact of the mutantOAS1R gene on promoting apoptosis under varying conditions. In oneembodiment, cell culture mutant forms of OAS1R can be assessed relativeto wild-type OAS1 sequences for their ability to promote apoptosis incells infected with a number of viruses including BVDV, HCV, and otherflaviviruses. As one skilled in the art will recognize, numerous methodsfor measuring apoptosis are available. The most common method involvesthe detection of the characteristic genomic “DNA laddering” effect inapoptosing cells using fluorescent conjugation methods coupled toagarose gel electrophoresis.

The ability of defective interfering viruses to potentiate the effectsof OAS1R mutant forms can be tested in cell culture and in small animalmodels.

The degree to which the presence or absence of OAS1R genotypes affectsother human phenotypes can also be examined. For instance, OAS1Rmutations are evaluated for their association with viral titer andspontaneous viral clearance in HCV infected subjects. Similar methods ofcorrelating host OAS1 genotype with the course of other flavivirusinfections can also be undertaken. The impact of OAS1R mutations onpromoting successful outcomes during interferon or interferon withribavirin treatment in HCV infected patients is also examined. Thesemutations may not only confer a level of infection resistance, but alsopromote spontaneous viral clearance in infected subjects with or withoutinterferon-ribavirin treatment. Furthermore, it has been reported thatschizophrenia occurs at a higher frequency in geographic areas that areendemic for flavivirus infection, suggesting an association betweenflavivirus resistance alleles and predisposition to schizophrenia. Thislink is evaluated by performing additional genetic association studiesinvolving the schizophrenia phenotype and the OAS1R mutations. Theimpact of OAS1R mutations on susceptibility to IDDM, prostate and othercancers, and schizophrenia will also be evaluated.

The invention discloses OAS1R variant mRNAs (identified as SEQ ID NO:36through SEQ ID NO:43) that are novel and have utility. The invention isnot limited by the mode of use of the disclosed variant mRNAs. In onepreferred embodiment, these variant mRNAs are used in differentiallyscreening human subjects for increased or decreased viral (includingHCV) susceptibility. In other preferred embodiments, these variant mRNAsare useful in screening for susceptibility to IDDM, prostate and othercancers, and/or schizophrenia. Such differential screening is performedby expression analyses known to those skilled in the art to determinerelative amounts of one or more variant OAS1R mRNAs present in samplesderived from a given human subject. Increased or decreased amounts ofone or more OAS1R mRNA variants in a human subject's sample relative toa control sample is indicative of the subject's degree of susceptibilityto viral, IDDM, prostate and other cancers, and/or schizophrenia, asappropriate to the test under consideration.

As discussed herein, 2′,5′-oligoadenylate synthetases (OAS) are a familyof IFN-α-inducible, RNA dependent effector molecules enzymes thatsynthesize short 2′ to 5′ linked oligoadenylate (2-5A) molecules fromATP. OAS enzymes constitute an important part of the nonspecific immunedefense against viral infections and have been used as a cellular markerfor viral infection. In addition to the role in hepatitis C infectiondiscussed herein, OAS activity is implicated in other disease states,particularly those in which a viral infection plays a role.

While specific pathogenic mechanisms are subjects of current analysis,viral infections are believed to play a role in the development ofdiseases such as diabetes. Lymphocytic OAS activity is significantlyelevated in patients with type 1 diabetes, suggesting that OAS may be animportant link between viral infections and disease development. In astudy involving diabetic twins from monozygotic twin pairs,Bonnevie-Nielsen et al. (Clin Immunol. 2000 July; 96(1): 11-8) showedthat OAS is persistently activated in both recent-onset andlong-standing type 1 diabetes. Continuously elevated OAS activity intype 1 diabetes is clearly different from a normal antiviral responseand might indicate a chronic stimulation of the enzyme, a failure ofdown regulatory mechanisms, or an aberrant response to endogenous orexogenous viruses or their products.

A more direct link between a viral infection and the development ofdiabetes is exemplified by a number of studies showing that between 13and 33% of patients with chronic hepatitis C have diabetes mellitus(type 2 diabetes), a level that is significantly increased compared withthat in matched healthy controls or patients with chronic hepatitis B(Knobler et al. Am J Gastroenterol. 2003 December; 98(12):2751-6). WhileOAS has not to date been reported to play a role in the development ofdiabetes mellitus following hepatitis C infection, it may be a usefulmarker for the antiviral response system. Furthermore, the resultsreported according to the present invention illustrate that if hepatitisC infection is causally related to diabetes mellitus, inhibition orabolition of hepatitis C infection using the compositions and methodsdisclosed herein may be advantageous in preventing or alleviatingdevelopment of diabetes mellitus.

A further published study has shown that OAS plays an essential role inwound healing and its pathological disorders, particularly in the caseof venous ulcers and diabetes-associated poorly-healing wounds (WO02/090552). In the case of poor wound healing, OAS mRNA levels in theaffected tissues were reduced, rather than elevated as in lymphocytesderived from patients suffering from type 1 diabetes. These findingspoint to OAS as an etiologically important marker of immune reactions indiabetes and diabetes-related wound healing.

OAS may also play an intermediary role in cell processes involved inprostate cancer. A primary biochemical function of OAS is to promote theactivity of RNaseL, a uniquely-regulated endoribonuclease that isenzymatically stimulated by 2-5A molecules. RNaseL has awell-established role in mediating the antiviral effects of IFN, and isa strong candidate for the hereditary prostate cancer 1 allele (HPC1).Mutations in RNaseL have been shown to predispose men to an increasedincidence of prostate cancer, which in some cases reflect moreaggressive disease and/or decreased age of onset compared with non RNaseL-linked cases. Xiang et al. (Cancer Res. 2003 Oct. 15; 63(20):6795-801)demonstrated that biostable phosphorothiolate analogs of 2-5A inducedRNaseL activity and caused apoptosis in cultures of late-stagemetastatic human prostate cancer cell lines. Their findings suggest thatthe elevation of OAS activity with a concurrent increase in 2-5A levelsmay facilitate the destruction of cancer cells through a potentapoptotic pathway. Thus, use of compositions and methods disclosedherein may find utility in the detection, treatment and/or prevention ofprostate cancer.

OAS may further play a role in normal cell growth regulation, eitherthrough its regulation of RNaseL or through another as yet undiscoveredpathway. There is considerable evidence to support the importance of OASin negatively regulating cell growth. Rysiecki et al. (J. InterferonRes. 1989 December; 9(6):649-57) demonstrated that stable transfectionof human OAS into a glioblastoma cell line results in reduced cellularproliferation. OAS levels have also been shown to be measurable inseveral studies comparing quiescent versus proliferating cell lines(e.g. Hassel and Ts'O, Mol Carcinog. 1992; 5(1):41-51 and Kimchi et al.,Eur J Biochem. 1981; 114(1):5-10) and in each case the OAS levels weregreatest in quiescent cells. Other studies have shown a correlationbetween OAS level and cell cycle phase, with OAS levels rising sharplyduring late S phase and then dropping abruptly in G2 (Wells andMallucci, Exp Cell Res. 1985 July; 159(1):27-36). Several studies haveshown a correlation between the induction of OAS and the onset ofantiproliferative effects following stimulation with various forms ofinterferon (see Player and Torrence, Pharmacol Ther. 1998 May;78(2):55-113). Induction of OAS has also been shown during celldifferentiation (e.g. Salzberg et al., J Cell Sci. 1996 June; 109(Pt6):1517-26 and Schwartz and Nilson, Mol Cell Biol. 1989 September;9(9):3897-903). Other reports of induction of OAS by platelet derivedgrowth factor (PDGF) (Zullo et al. Cell. 1985 December; 43(3 Pt2):793-800) and under conditions of heat-shock induced growth(Chousternan et al., J Biol Chem. 1987 Apr. 5; 262(10):4806-11) lead tothe hypothesis that induction of OAS is a normal cell growth controlmechanism. Thus, use of compositions and methods disclosed herein mayfind broad utility in the detection, treatment and/or prevention ofcancer.

Polynucleotide Analysis

An oligoadenylate synthetase gene is a nucleic acid whose nucleotidesequence codes for oligoadenylate synthetase, mutant oligoadenylatesynthetase, or oligoadenylate synthetase pseudogene. It can be in theform of genomic DNA, an mRNA or cDNA, and in single or double strandedform. Preferably, genomic DNA is used because of its relative stabilityin biological samples compared to mRNA. The sequence of a polynucleotideconsisting of consecutive nucleotides 2,130,000-2,157,999 of thecomplete genomic sequence of the reference oligoadenylate synthetasegene is provided in the Sequence Listing as SEQ ID NO:19, andcorresponds to Genbank Accession No. NT_(—)009775.13.

The nucleic acid sample is obtained from cells, typically peripheralblood leukocytes. Where mRNA is used, the cells are lysed under RNaseinhibiting conditions. In one embodiment, the first step is to isolatethe total cellular mRNA. Poly A+ mRNA can then be selected byhybridization to an oligo-dT cellulose column.

In preferred embodiments, the nucleic acid sample is enriched for apresence of oligoadenylate synthetase allelic material. Enrichment istypically accomplished by subjecting the genomic DNA or mRNA to a primerextension reaction employing a polynucleotide synthesis primer asdescribed herein. Particularly preferred methods for producing a sampleto be assayed use preselected polynucleotides as primers in a polymerasechain reaction (PCR) to form an amplified (PCR) product.

Preparation of Polynucleotide Primers

The term “polynucleotide” as used herein in reference to primers, probesand nucleic acid fragments or segments to be synthesized by primerextension is defined as a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three. Itsexact size will depend on many factors, which in turn depends on theultimate conditions of use.

The term “primer” as used herein refers to a polynucleotide whetherpurified from a nucleic acid restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is induced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, reverse transcriptase and thelike, and at a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency, but may alternatively be indouble stranded form. If double stranded, the primer is first treated toseparate it from its complementary strand before being used to prepareextension products. Preferably, the primer is a polydeoxyribonucleotide.The primer must be sufficiently long to prime the synthesis of extensionproducts in the presence of the agents for polymerization. The exactlengths of the primers will depend on many factors, includingtemperature and the source of primer. For example, depending on thecomplexity of the target sequence, a polynucleotide primer typicallycontains 15 to 25 or more nucleotides, although it can contain fewernucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with template.

The primers used herein are selected to be “substantially” complementaryto the different strands of each specific sequence to be synthesized oramplified. This means that the primer must be sufficiently complementaryto non-randomly hybridize with its respective template strand.Therefore, the primer sequence may or may not reflect the exact sequenceof the template. For example, a non-complementary nucleotide fragmentcan be attached to the 5′ end of the primer, with the remainder of theprimer sequence being substantially complementary to the strand. Suchnon-complementary fragments typically code for an endonucleaserestriction site. Alternatively, non-complementary bases or longersequences can be interspersed into the primer, provided the primersequence has sufficient complementarity with the sequence of the strandto be synthesized or amplified to non-randomly hybridize therewith andthereby form an extension product under polynucleotide synthesizingconditions.

Primers of the present invention may also contain a DNA-dependent RNApolymerase promoter sequence or its complement. See for example, Krieg,et al., Nucl. Acids Res., 12:7057-70 (1984); Studier, et al., J. Mol.Biol., 189:113-130 (1986); and Molecular Cloning: A Laboratory Manual,Second Edition, Maniatis, et al., eds., Cold Spring Harbor, N.Y. (1989).

When a primer containing a DNA-dependent RNA polymerase promoter isused, the primer is hybridized to the polynucleotide strand to beamplified and the second polynucleotide strand of the DNA-dependent RNApolymerase promoter is completed using an inducing agent such as E. coliDNA polymerase I, or the Klenow fragment of E. coli DNA polymerase. Thestarting polynucleotide is amplified by alternating between theproduction of an RNA polynucleotide and DNA polynucleotide.

Primers may also contain a template sequence or replication initiationsite for a RNA-directed RNA polymerase. Typical RNA-directed RNApolymerase include the QB replicase described by Lizardi, et al.,Biotechnology, 6:1197-1202 1988). RNA-directed polymerases produce largenumbers of RNA strands from a small number of template RNA strands thatcontain a template sequence or replication initiation site. Thesepolymerases typically give a one million-fold amplification of thetemplate strand as has been described by Kramer, et al., J. Mol. Biol.,89:719-736 (1974).

The polynucleotide primers can be prepared using any suitable method,such as, for example, the phosphotriester or phosphodiester methods seeNarang, et al., Meth. Enzymol., 68:90, (1979); U.S. Pat. Nos. 4,356,270,4,458,066, 4,416,988, 4,293,652; and Brown, et al., Meth. Enzymol.,68:109 (1979).

The choice of a primer's nucleotide sequence depends on factors such asthe distance on the nucleic acid from the hybridization point to theregion coding for the mutation to be detected, its hybridization site onthe nucleic acid relative to any second primer to be used, and the like.

If the nucleic acid sample is to be enriched for oligoadenylatesynthetase gene material by PCR amplification, two primers, i.e., a PCRprimer pair, must be used for each coding strand of nucleic acid to beamplified. The first primer becomes part of the non-coding (anti-senseor minus or complementary) strand and hybridizes to a nucleotidesequence on the plus or coding strand. Second primers become part of thecoding (sense or plus) strand and hybridize to a nucleotide sequence onthe minus or non-coding strand. One or both of the first and secondprimers can contain a nucleotide sequence defining an endonucleaserecognition site. The site can be heterologous to the oligoadenylatesynthetase gene being amplified.

In one embodiment, the present invention utilizes a set ofpolynucleotides that form primers having a priming region located at the3′-terminus of the primer. The priming region is typically the 3′-most(3′-terminal) 15 to 30 nucleotide bases. The 3′-terminal priming portionof each primer is capable of acting as a primer to catalyze nucleic acidsynthesis, i.e., initiate a primer extension reaction off its 3′terminus. One or both of the primers can additionally contain a5′-terminal (5′-most) non-priming portion, i.e., a region that does notparticipate in hybridization to the preferred template.

In PCR, each primer works in combination with a second primer to amplifya target nucleic acid sequence. The choice of PCR primer pairs for usein PCR is governed by considerations as discussed herein for producingoligoadenylate synthetase gene regions. When a primer sequence is chosento hybridize (anneal) to a target sequence within an oligoadenylatesynthetase gene allele intron, the target sequence should be conservedamong the alleles in order to insure generation of target sequence to beassayed.

Polymerase Chain Reaction

Oligoadenylate synthetase genes are comprised of polynucleotide codingstrands, such as mRNA and/or the sense strand of genomic DNA. If thegenetic material to be assayed is in the form of double stranded genomicDNA, it is usually first denatured, typically by melting, into singlestrands. The nucleic acid is subjected to a PCR reaction by treating(contacting) the sample with a PCR primer pair, each member of the pairhaving a preselected nucleotide sequence. The PCR primer pair is capableof initiating primer extension reactions by hybridizing to nucleotidesequences, preferably at least about 10 nucleotides in length, morepreferably at least about 20 nucleotides in length, conserved within theoligoadenylate synthetase alleles. The first primer of a PCR primer pairis sometimes referred to herein as the “anti-sense primer” because ithybridizes to a non-coding or anti-sense strand of a nucleic acid, i.e.,a strand complementary to a coding strand. The second primer of a PCRprimer pair is sometimes referred to herein as the “sense primer”because it hybridizes to the coding or sense strand of a nucleic acid.

The PCR reaction is performed by mixing the PCR primer pair, preferablya predetermined amount thereof, with the nucleic acids of the sample,preferably a predetermined amount thereof, in a PCR buffer to form a PCRreaction admixture. The admixture is thermocycled for a number ofcycles, which is typically predetermined, sufficient for the formationof a PCR reaction product, thereby enriching the sample to be assayedfor oligoadenylate synthetase genetic material.

PCR is typically carried out by thermocycling i.e., repeatedlyincreasing and decreasing the temperature of a PCR reaction admixturewithin a temperature range whose lower limit is about 30 degrees Celsius(30° C.) to about 5° C. and whose upper limit is about 90° C. to about10° C. The increasing and decreasing can be continuous, but ispreferably phasic with time periods of relative temperature stability ateach of temperatures favoring polynucleotide synthesis, denaturation andhybridization.

A plurality of first primer and/or a plurality of second primers can beused in each amplification, e.g., one species of first primer can bepaired with a number of different second primers to form severaldifferent primer pairs. Alternatively, an individual pair of first andsecond primers can be used. In any case, the amplification products ofamplifications using the same or different combinations of first andsecond primers can be combined for assaying for mutations.

The PCR reaction is performed using any suitable method. Generally itoccurs in a buffered aqueous solution, i.e., a PCR buffer, preferably ata pH of 7-9, most preferably about 8. Preferably, a molar excess (forgenomic nucleic acid, usually about 10⁶:1 primer:template) of the primeris admixed to the buffer containing the template strand. A large molarexcess is preferred to improve the efficiency of the process.

The PCR buffer also contains the deoxyribonucleotide triphosphates(polynucleotide synthesis substrates) dATP, dCTP, dGTP, and dTTP and apolymerase, typically thermostable, all in adequate amounts for primerextension (polynucleotide synthesis) reaction. The resulting solution(PCR admixture) is heated to about 90° C.-100° C. for about 1 to 10minutes, preferably from 1 to 4 minutes. After this heating period thesolution is allowed to cool to 54° C., which is preferable for primerhybridization. The synthesis reaction may occur at from room temperatureup to a temperature above which the polymerase (inducing agent) nolonger functions efficiently. The thermocycling is repeated until thedesired amount of PCR product is produced. An exemplary PCR buffercomprises the following: 50 mM KCl; 10 mM Tris-HCl at pH 8.3; 1.5 mMMgCl.; 0.001% (wt/vol) gelatin, 200 μM DATP; 200 μM dTTP; 200 μM dCTP;200² μM dGTP; and 2.5 units Thermus aquaticus (Taq) DNA polymerase I(U.S. Pat. No. 4,889,818) per 100 microliters of buffer.

The inducing agent may be any compound or system which will function toaccomplish the synthesis of primer extension products, includingenzymes. Suitable enzymes for this purpose include, for example, E. coliDNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, reverse transcriptase, andother enzymes, including heat-stable enzymes, which will facilitatecombination of the nucleotides in the proper manner to form the primerextension products which are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be inducing agents, however, which initiate synthesis at the 5′ endand proceed in the above direction, using the same process as describedabove.

The inducing agent also may be a compound or system which will functionto accomplish the synthesis of RNA primer extension products, includingenzymes. In preferred embodiments, the inducing agent may be aDNA-dependent RNA polymerase such as T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase. These polymerases produce acomplementary RNA polynucleotide. The high turn-over rate of the RNApolymerase amplifies the starting polynucleotide as has been describedby Chamberlin, et al., The Enzymes, ed. P. Boyer, pp. 87-108, AcademicPress, New York (1982). Amplification systems based on transcriptionhave been described by Gingeras, et al., in PCR Protocols, A Guide toMethods and Applications, pp. 245-252, Innis, et al., eds, AcademicPress, Inc., San Diego, Calif. (1990).

If the inducing agent is a DNA-dependent RNA polymerase and, thereforeincorporates ribonucleotide triphosphates, sufficient amounts of ATP,CTP, GTP and UTP are admixed to the primer extension reaction admixtureand the resulting solution is treated as described above.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the process.

The PCR reaction can advantageously be used to incorporate into theproduct a preselected restriction site useful in detecting a mutation inthe oligoadenylate synthetase gene.

PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in severaltexts including PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, New York (1989); and PCRProtocols: A Guide to Methods and Applications, Innis, et al., eds.,Academic Press, San Diego, Calif. (1990).

In some embodiments, two pairs of first and second primers are used peramplification reaction. The amplification reaction products obtainedfrom a plurality of different amplifications, each using a plurality ofdifferent primer pairs, can be combined or assayed separately.

However, the present invention contemplates amplification using only onepair of first and second primers. Exemplary primers for amplifying thesections of DNA containing the mutations disclosed herein are shownbelow in Table 1. AmpliconA corresponds to the polynucleotide sequencethat contains the mutations referred to in SEQ ID NO:1-3. AmpliconBcorresponds to the polynucleotide sequence containing the mutationsreferred to in SEQ ID NO:4-7 and SEQ ID NO:60. AmpliconC corresponds tothe polynucleotide sequence containing the mutation referred to in SEQID NO:57. AmpliconD corresponds to the polynucleotide sequencecontaining the mutation referred to in SEQ ID NO:58. AmpliconEcorresponds to the polynucleotide sequence containing the mutationreferred to in SEQ ID NO: 59. AmpliconF corresponds to thepolynucleotide sequence containing the mutation referred to in SEQ IDNO:61. AmpliconG corresponds to the polynucleotide sequences containingthe mutation referred to in SEQ ID NO: 62-64.

TABLE 1 Amplicons Containing Mutations of the Present Invention Productsize Amplicon PrimerA PrimerB (bp) AmpliconA 5′-AATGGACCTCAAGACTTCCC-3′5′-ATTCTCCCTTCTGTTGCAGG-3′ 509 (SEQ ID NO: 8) (SEQ ID NO: 9) AmpliconB5′-TCCAGATGGCATGTCACAGT-3′ 5′-GAGCTATGCTTGGCACATAG-3′ 747 (SEQ ID NO:10) (SEQ ID NO: 11) AmpliconC 5′-CACAAGAGTGAACCTTAATGT-3′5′-CCAGGAAGTGGAAAGATCAT-3′ 603 (SEQ ID NO: 65) (SEQ ID NO: 66) AmpliconD5′-ATCTCCCACAGTTTGAGAGC-3′ 5′-TCAGCCTCCAAAAGTGTTGG-3′ 553 (SEQ ID NO:67) (SEQ ID NO: 68) AmpliconE 5′-GGGTACATGTGCACAATGTG-3′5′-CCCTTATACAAAATTCAACTC-3′ 532 (SEQ ID NO: 69) (SEQ ID NO: 70)AmpliconF 5′-GAGCCAAGAAGTACAGATGC-3′ 5′-AGGACAGAGCTGTCCAATAG-3′ 648 (SEQID NO: 71) (SEQ ID NO: 72) AmpliconG 5′-GGCTCAGAGAAGCTAAGTGA-3′5′-CCACAGCATCCTTTTCAGTC-3′ 581 (SEQ ID NO: 73) (SEQ ID NO: 74)Table 2 discloses the position in the above Amplicons of the mutationsof the invention.

TABLE 2 Position of Mutations of the Invention in Amplicons Position inAmplicon (relative to 5′ end of Mutation Amplicon PrimerA side ofAmplicon) 1 (SEQ ID NO:1) Amplicon A 134 2 (SEQ ID NO:2) Amplicon A 1553 (SEQ ID NO:3) Amplicon A 384 4 (SEQ ID NO:4) Amplicon B 98 5 (SEQ IDNO:5) Amplicon B 114 6 (SEQ ID NO:6) Amplicon B 142 7 (SEQ ID NO:7)Amplicon B 347  8 (SEQ ID NO:57) Amplicon C 319  9 (SEQ ID NO:58)Amplicon D 404 10 (SEQ ID NO:59) Amplicon E 133 11 (SEQ ID NO:60)Amplicon B 320 12 (SEQ ID NO:61) Amplicon F 367 13 (SEQ ID NO:62)Amplicon G 138 14 (SEQ ID NO:63) Amplicon G 210 15 (SEQ ID NO:64)Amplicon G 253

Nucleic Acid Sequence Analysis

Nucleic acid sequence analysis is approached by a combination of (a)physiochemical techniques, based on the hybridization or denaturation ofa probe strand plus its complementary target, and (b) enzymaticreactions with endonucleases, ligases, and polymerases. Nucleic acid canbe assayed at the DNA or RNA level. The former analyzes the geneticpotential of individual humans and the latter the expressed informationof particular cells.

In assays using nucleic acid hybridization, detecting the presence of aDNA duplex in a process of the present invention can be accomplished bya variety of means.

In one approach for detecting the presence of a DNA duplex, anoligonucleotide that is hybridized in the DNA duplex includes a label orindicating group that will render the duplex detectable. Typically suchlabels include radioactive atoms, chemically modified nucleotide bases,and the like.

The oligonucleotide can be labeled, i.e., operatively linked to anindicating means or group, and used to detect the presence of a specificnucleotide sequence in a target template.

Radioactive elements operatively linked to or present as part of anoligonucleotide probe (labeled oligonucleotide) provide a useful meansto facilitate the detection of a DNA duplex. A typical radioactiveelement is one that produces beta ray emissions. Elements that emit betarays, such as ³H, ¹²C, ³²P and ³⁵S represent a class of beta rayemission-producing radioactive element labels. A radioactivepolynucleotide probe is typically prepared by enzymatic incorporation ofradioactively labeled nucleotides into a nucleic acid using DNA kinase.

Alternatives to radioactively labeled oligonucleotides areoligonucleotides that are chemically modified to contain metalcomplexing agents, biotin-containing groups, fluorescent compounds, andthe like.

One useful metal complexing agent is a lanthanide chelate formed by alanthanide and an aromatic beta-diketone, the lanthanide being bound tothe nucleic acid or oligonucleotide via a chelate-forming compound suchas an EDTA-analogue so that a fluorescent lanthanide complex is formed.See U.S. Pat. Nos. 4,374,120, 4,569,790 and published Patent ApplicationEP0139675 and WO87/02708.

Biotin or acridine ester-labeled oligonucleotides and their use to labelpolynucleotides have been described. See U.S. Pat. No. 4,707,404,published Patent Application EP0212951 and European Patent No. 0087636.Useful fluorescent marker compounds include fluorescein, rhodamine,Texas Red, NBD and the like.

A labeled oligonucleotide present in a DNA duplex renders the duplexitself labeled and therefore distinguishable over other nucleic acidspresent in a sample to be assayed. Detecting the presence of the labelin the duplex and thereby the presence of the duplex, typically involvesseparating the DNA duplex from any labeled oligonucleotide probe that isnot hybridized to a DNA duplex.

Techniques for the separation of single stranded oligonucleotide, suchas non-hybridized labeled oligonucleotide probe, from DNA duplex arewell known, and typically involve the separation of single stranded fromdouble stranded nucleic acids on the basis of their chemical properties.More often separation techniques involve the use of a heterogeneoushybridization format in which the non-hybridized probe is separated,typically by washing, from the DNA duplex that is bound to an insolublematrix. Exemplary is the Southern blot technique, in which the matrix isa nitrocellulose sheet and the label is ³²P. Southern, J. Mol. Biol.,98:503 (1975).

The oligonucleotides can also be advantageously linked, typically at ornear their 5′-terminus, to a solid matrix, i.e., aqueous insoluble solidsupport. Useful solid matrices are well known in the art and includecross-linked dextran such as that available under the tradename SEPHADEXfrom Pharmacia Fine Chemicals (Piscataway, N.J.); agarose, polystyreneor latex beads about 1 micron to about 5 millimeters in diameter,polyvinyl chloride, polystyrene, cross-linked polyacrylamide,nitrocellulose or nylon-based webs such as sheets, strips, paddles,plates microtiter plate wells and the like.

It is also possible to add “linking” nucleotides to the 5′ or 3′ end ofthe member oligonucleotide, and use the linking oligonucleotide tooperatively link the member to the solid support.

In nucleotide hybridizing assays, the hybridization reaction mixture ismaintained in the contemplated method under hybridizing conditions for atime period sufficient for the oligonucleotides having complementarityto the predetermined sequence on the template to hybridize tocomplementary nucleic acid sequences present in the template to form ahybridization product, i.e., a complex containing oligonucleotide andtarget nucleic acid.

The phrase “hybridizing conditions” and its grammatical equivalents,when used with a maintenance time period, indicates subjecting thehybridization reaction admixture, in the context of the concentrationsof reactants and accompanying reagents in the admixture, to time,temperature and pH conditions sufficient to allow one or moreoligonucleotides to anneal with the target sequence, to form a nucleicacid duplex. Such time, temperature and pH conditions required toaccomplish hybridization depend, as is well known in the art, on thelength of the oligonucleotide to be hybridized, the degree ofcomplementarity between the oligonucleotide and the target, the guanineand cytosine content of the oligonucleotide, the stringency ofhybridization desired, and the presence of salts or additional reagentsin the hybridization reaction admixture as may affect the kinetics ofhybridization. Methods for optimizing hybridization conditions for agiven hybridization reaction admixture are well known in the art.

Typical hybridizing conditions include the use of solutions buffered topH values between 4 and 9, and are carried out at temperatures from 4°C. to 37° C., preferably about 12° C. to about 30° C., more preferablyabout 22° C., and for time periods from 0.5 seconds to 24 hours,preferably 2 minutes (min) to 1 hour.

Hybridization can be carried out in a homogeneous or heterogeneousformat as is well known. The homogeneous hybridization reaction occursentirely in solution, in which both the oligonucleotide and the nucleicacid sequences to be hybridized (target) are present in soluble forms insolution. A heterogeneous reaction involves the use of a matrix that isinsoluble in the reaction medium to which either the oligonucleotide,polynucleotide probe or target nucleic acid is bound.

Where the nucleic acid containing a target sequence is in a doublestranded (ds) form, it is preferred to first denature the dsDNA, as byheating or alkali treatment, prior to conducting the hybridizationreaction. The denaturation of the dsDNA can be carried out prior toadmixture with an oligonucleotide to be hybridized, or can be carriedout after the admixture of the dsDNA with the oligonucleotide.

Predetermined complementarity between the oligonucleotide and thetemplate is achieved in two alternative manners. A sequence in thetemplate DNA may be known, such as where the primer to be formed canhybridize to known oligoadenylate synthetase sequences and can initiateprimer extension into a region of DNA for sequencing purposes, as wellas subsequent assaying purposes as described herein, or where previoussequencing has determined a region of nucleotide sequence and the primeris designed to extend from the recently sequenced region into a regionof unknown sequence. This latter process has been referred to a“directed sequencing” because each round of sequencing is directed by aprimer designed based on the previously determined sequence.

Effective amounts of the oligonucleotide present in the hybridizationreaction admixture are generally well known and are typically expressedin terms of molar ratios between the oligonucleotide to be hybridizedand the template. Preferred ratios are hybridization reaction mixturescontaining equimolar amounts of the target sequence and theoligonucleotide. As is well known, deviations from equal molarity willproduce hybridization reaction products, although at lower efficiency.Thus, although ratios where one component can be in as much as 100 foldmolar excess relative to the other component, excesses of less than 50fold, preferably less than 10 fold, and more preferably less than twofold are desirable in practicing the invention.

Detection of Membrane-Immobilized Target Sequences

In the DNA (Southern) blot technique, DNA is prepared by PCRamplification as previously discussed. The PCR products (DNA fragments)are separated according to size in an agarose gel and transferred(blotted) onto a nitrocellulose or nylon membrane. Conventionalelectrophoresis separates fragments ranging from 100 to 30,000 basepairs while pulsed field gel electrophoresis resolves fragments up to 20million base pairs in length. The location on the membrane a containingparticular PCR product is determined by hybridization with a specific,labeled nucleic acid probe.

In preferred embodiments, PCR products are directly immobilized onto asolid-matrix (nitrocellulose membrane) using a dot-blot (slot-blot)apparatus, and analyzed by probe-hybridization. See U.S. Pat. Nos.4,582,789 and 4,617,261.

Immobilized DNA sequences may be analyzed by probing withallele-specific oligonucleotide (ASO) probes, which are synthetic DNAnoligomers of approximately 15, 17, 20, 25 or up to about 30 nucleotidesin length. These probes are long enough to represent unique sequences inthe genome, but sufficiently short to be destabilized by an internalmismatch in their hybridization to a target molecule. Thus, anysequences differing at single nucleotides may be distinguished by thedifferent denaturation behaviors of hybrids between the ASO probe andnormal or mutant targets under carefully controlled hybridizationconditions. Exemplary probes are disclosed herein as SEQ ID NO:1-7 andSEQ ID NO:57-64 (Table 3), but any probes are suitable as long as theyhybridize specifically to the region of the OAS1 gene carrying the pointmutation of choice, and are capable of specifically distinguishingbetween polynucleotide carrying the point mutation and a wild typepolynucleotide.

Detection of Target Sequences in Solution

Several rapid techniques that do not require nucleic acid purificationor immobilization have been developed. For example, probe/target hybridsmay be selectively isolated on a solid matrix, such as hydroxylapatite,which preferentially binds double-stranded nucleic acids. Alternatively,probe nucleic acids may be immobilized on a solid support and used tocapture target sequences from solution. Detection of the targetsequences can be accomplished with the aid of a second, labeled probethat is either displaced from the support by the target sequence in acompetition-type assay or joined to the support via the bridging actionof the target sequence in a sandwich-type format.

In the oligonucleotide ligation assay (OLA), the enzyme DNA ligase isused to covalently join two synthetic oligonucleotide sequences selectedso that they can base pair with a target sequence in exact head-to-tailjuxtaposition. Ligation of the two oligomers is prevented by thepresence of mismatched nucleotides at the junction region. Thisprocedure allows for the distinction between known sequence variants insamples of cells without the need for DNA purification. The joining ofthe two oligonucleotides may be monitored by immobilizing one of the twooligonucleotides and observing whether the second, labeledoligonucleotide is also captured.

Scanning Techniques for Detection of Base Substitutions

Three techniques permit the analysis of probe/target duplexes severalhundred base pairs in length for unknown single-nucleotide substitutionsor other sequence differences. In the ribonuclease (RNase) A technique,the enzyme cleaves a labeled RNA probe at positions where it ismismatched to a target RNA or DNA sequence. The fragments may beseparated according to size allowing for the determination of theapproximate position of the mutation. See U.S. Pat. No. 4,946,773.

In the denaturing gradient gel technique, a probe-target DNA duplex isanalyzed by electrophoresis in a denaturing gradient of increasingstrength. Denaturation is accompanied by a decrease in migration rate. Aduplex with a mismatched base pair denatures more rapidly than aperfectly matched duplex.

A third method relies on chemical cleavage of mismatched base pairs. Amismatch between T and C, G, or T, as well as mismatches between C andT, A, or C, can be detected in heteroduplexes. Reaction with osmiumtetroxide (T and C mismatches) or hydroxylamine (C mismatches) followedby treatment with piperidine cleaves the probe at the appropriatemismatch.

Therapeutic Agents for Restoring and/or Enhancing OAS1 Function

Where a mutation in the OAS1 gene leads to defective OAS1 function andthis defective function is associated with increased susceptibility of apatient to pathogenic infection, whether through lower levels of OAS1protein, mutation in the protein affecting its function, or othermechanisms, it may be advantageous to treat the patient with wild typeOAS1 protein. Furthermore, if the mutation gives rise ininfection-resistant carriers to a form of the protein that differs fromthe wild-type protein, and that has an advantage in terms of inhibitingHCV infection, it may be advantageous to administer a protein encoded bythe mutated gene. As described previously, administration of eithernative or mutant forms of OAS1 proteins or polypeptides may also beadvantageous in the treatment of other indications including but notlimited to cancer, diabetes mellitus, and wound healing. The discussionbelow pertains to administration of any of the foregoing proteins orpolypeptides.

The polypeptides of the present invention, including those encoded byOAS1R, may be a naturally purified product, or a product of chemicalsynthetic procedures, or produced by recombinant techniques from aprokaryotic or eukaryotic host (for example, by bacterial, yeast, higherplant, insect and mammalian cells in culture) of a polynucleotidesequence of the present invention. Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated with mammalian or other eukaryoticcarbohydrates or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue (at positionminus 1).

The polypeptides of the present invention also include the proteinsequences defined in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 46, and SEQ ID NO: 47 and derivativesthereof. In addition to naturally occurring allelic forms of thepolypeptide(s) the present invention also embraces analogs and fragmentsthereof, which function similarly to the naturally occurring allelicforms. Thus, for example, one or more of the amino acid residues of thepolypeptide may be replaced by conserved amino acid residues, as long asthe function of the OAS1R protein is maintained. Examples 8-10, below,provide representative illustrations of suitable amino acid replacementswith regard to the polypeptides of the present invention. As anotherexample, the polypeptides of the present invention specifically includethe truncated or analog forms of OAS1R defined in SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO:52. As discussedpreviously, SEQ ID NO: 51 represents the shortened form of OAS1possessed by chimpanzees and SEQ ID NO: 52 represents acarboxyl-terminus fragment of the longer but still truncated formpossessed by gorillas. SEQ ID NO: 49 and SEQ ID NO: 50 representsynthetic human OAS1R constructs truncated to the correspondingchimpanzee and gorilla sites of truncation, respectively. SEQ ID NO: 48represents a synthetic human OAS1R polypeptide truncated to a lengthintermediate to the chimpanzee and gorilla forms. SEQ ID NO: 48 hasfurther been demonstrated to be enzymatically active by methods known inthe art as disclosed elsewhere herein. Correspondingly, the remaininghighly similar truncated forms may also be demonstrated to beenzymatically active. As those skilled in the art will appreciate,therapeutic use of truncated but functional forms of OAS1R polypeptidescan preclude the development of antibody response which would otherwisehinder the therapeutic efficacy of the polypeptide. The foregoingtruncated polypeptides, and others that can be envisioned by one skilledin the art, maintain function but remove non-ubiquitous portions of thepolypeptide that could induce antibody response in individuals notpossessing the full length OAS1R polypeptide endogenously. Those skilledin the art will also appreciate that smaller polypeptides, in general,are more amenable to the complexities of manufacturing, delivery, andclearance typically encountered in therapeutic development.Additionally, those skilled in the art will appreciate that theoccurrence of distinct homozygous truncating variants in chimpanzee andgorilla are also highly suggestive for the broad anti-viral potency ofthe presently disclosed truncated OAS1 forms. Although the truncatedpolypeptide forms specifically disclosed above represent truncations tothe carboxyl-terminus of the polypeptide, the invention is not limitedby the form of the fragment and specifically includes amino-terminustruncations and internal amino acid deletions that retain enzymaticfunction.

Also included in the scope of the invention are polypeptides that retainat least one activity of a specific disclosed polypeptide, but differfrom the disclosed amino acid sequence. Such polypeptides preferablyhave at least 80% sequence homology, preferably 85% sequence homology,more preferably 90% sequence homology, most preferably 95% more sequencehomology to the corresponding disclosed SEQ ID NO: as calculated usingstandard methods of alignment.

The polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as gene therapy. Thus, for example, cells may be transducedwith a polynucleotide (DNA or RNA) encoding the polypeptides ex vivowith those transduced cells then being provided to a patient to betreated with the polypeptide. Such methods are well known in the art.For example, cells may be transduced by procedures known in the art byuse of a retroviral particle containing RNA encoding the polypeptide ofthe present invention.

Similarly, transduction of cells may be accomplished in vivo forexpression of the polypeptide in vivo, for example, by procedures knownin the art. As known in the art, a producer cell for producing aretroviral particle containing RNA encoding the polypeptides of thepresent invention may be administered to a patient for transduction invivo and expression of the polypeptides in vivo.

These and other methods for administering the polypeptides of thepresent invention by such methods should be apparent to those skilled inthe art from the teachings of the present invention. For example, theexpression vehicle for transducing cells may be other than a retrovirus,for example, an adenovirus which may be used to transduce cells in vivoafter combination with a suitable delivery vehicle.

In the case where the polypeptides are prepared as a liquid formulationand administered by injection, preferably the solution is an isotonicsalt solution containing 140 millimolar sodium chloride and 10millimolar calcium at pH 7.4. The injection may be administered, forexample, in a therapeutically effective amount, preferably in a dose ofabout 1 μg/kg body weight to about 5 mg/kg body weight daily, takinginto account the routes of administration, health of the patient, etc.

The polypeptide(s) of the present invention may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the protein, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The polypeptide(s) of the present invention can also be modified bychemically linking the polypeptide to one or more moieties or conjugatesto enhance the activity, cellular distribution, or cellular uptake ofthe polypeptide(s). Such moieties or conjugates include lipids such ascholesterol, cholic acid, thioether, aliphatic chains, phospholipids andtheir derivatives, polyamines, polyethylene glycol (PEG), palmitylmoieties, and others as disclosed in, for example, U.S. Pat. Nos.5,514,758, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371,5,597,696 and 5,958,773.

The polypeptide(s) of the present invention may also be modified totarget specific cell types for a particular disease indication,including but not limited to liver cells in the case of hepatitis Cinfection. As can be appreciated by those skilled in the art, suitablemethods have been described that achieve the described targeting goalsand include, without limitation, liposomal targeting, receptor-mediatedendocytosis, and antibody-antigen binding. In one embodiment, theasiaglycoprotein receptor may be used to target liver cells by theaddition of a galactose moiety to the polypeptide(s). In anotherembodiment, mannose moieties may be conjugated to the polypeptide(s) inorder to target the mannose receptor found on macrophages and livercells. The polypeptide(s) of the present invention may also be modifiedfor cytosolic delivery by methods known to those skilled in the art,including, but not limited to, endosome escape mechanisms or proteintransduction domain (PTD) systems. PTD systems are disclosed in, forexample, Vives E, et al. (1997) J. Biol. Chem. 272: 16010-16017,Derossi, et al. (1994) J. Biol. Chem. 269: 10444-10450, Elliott, G etal. (1997) Cell 88:223-233, Wadia, J S et al. (2004) Nat. Med.10:310-315, and Kabouridis, P S. (2003) Trends Biotech., 21: 498-503.Known endosome escape systems include the use of ph-responsive polymericcarriers such as poly(propylacrylic acid). Known PTD systems range fromnatural peptides such as HIV-1 TAT, HSV-1 VP22, Drosophila Antennapedia,or diphtheria toxin to synthetic peptide carriers (Wadia and Dowdy, Cur.Opin. Biotech. 13:52-56, 2002; Becker-Hapak et. al., Methods 24:247-256,2001). FIG. 10 provides detailed description of several of theseexemplary PTDs. As one skilled in the art will recognize, multipledelivery and targeting methods may be combined. For example, thepolypeptide(s) of the present invention may be targeted to liver cellsby encapsulation within liposomes, such liposomes being conjugated togalactose for targeting to the asialoglycoprotein receptor.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptide of the present invention may be employed in conjunction withother therapeutic compounds.

When the OAS1 variants of the present invention are used as apharmaceutical, they can be given to mammals, in a suitable vehicle.When the polypeptides of the present invention are used as apharmaceutical as described above, they are given, for example, intherapeutically effective doses of about 10 μg/kg body weight to about 4mg/kg body weight daily, taking into account the routes ofadministration, health of the patient, etc. The amount given ispreferably adequate to achieve prevention or inhibition of infection bya virus, preferably a flavivirus, most preferably HCV, thus replicatingthe natural resistance found in humans carrying an OAS1R allele asdisclosed herein.

Inhibitor-based drug therapies that mimic the beneficial effects of atleast one mutation at position 2135728, 2135749, 2135978, 2144072,2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985,2156523, 2156595, or 2156638 of NT_(—)009775.13 are also envisioned, asdiscussed in detail below. As discussed previously, one exemplaryrationale for developing such inhibitors is the case where thebeneficial mutation diminishes or eradicates expression, translation, orfunction of one or more particular isoforms of OAS1. The presentinvention is not limited by the precise form or effect of the beneficialmutation nor the biological activity of the particular isoforms therebyaffected. In such case, one skilled in the art will appreciate theutility of therapeutically inhibiting said particular isoform(s) ofOAS1. These inhibitor-based therapies can take the form of chemicalentities, peptides or proteins, antisense oligonucleotides, smallinterference RNAs, and antibodies.

The proteins, their fragments or other derivatives, or analogs thereof,or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal,monoclonal, chimeric, single chain, Fab fragments, or the product of anFab expression library. Various procedures known in the art may be usedfor the production of polyclonal antibodies.

Antibodies generated against the polypeptide encoded by OAS1R of thepresent invention can be obtained by direct injection of the polypeptideinto an animal or by administering the polypeptide to an animal,preferably a nonhuman. The antibody so obtained will then bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies bindingthe whole native polypeptide. Moreover, a panel of such antibodies,specific to a large number of polypeptides, can be used to identify anddifferentiate such tissue. As an example, FIG. 9 demonstratesdevelopment of antibodies specific to particular exemplary polypeptidesof the present invention.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-597), the trioma technique, the human B-cell hybridomatechnique (Kozbor, et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Coe, etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

The antibodies can be used in methods relating to the localization andactivity of the protein sequences of the invention, e.g., for imagingthese proteins, measuring levels thereof in appropriate physiologicalsamples, and the like.

The present invention provides detectably labeled oligonucleotides forimaging OAS1 polynucleotides within a cell. Such oligonucleotides areuseful for determining if gene amplification has occurred, and forassaying the expression levels in a cell or tissue using, for example,in situ hybridization as is known in the art.

Therapeutic Agents for Inhibition of OAS1 Function

The present invention also relates to antisense oligonucleotidesdesigned to interfere with the normal function of OAS1 polynucleotides.Any modifications or variations of the antisense molecule which areknown in the art to be broadly applicable to antisense technology areincluded within the scope of the invention. Such modifications includepreparation of phosphorus-containing linkages as disclosed in U.S. Pat.Nos. 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361,5,625,050 and 5,958,773.

The antisense compounds of the invention can include modified bases asdisclosed in 5,958,773 and patents disclosed therein. The antisenseoligonucleotides of the invention can also be modified by chemicallylinking the oligonucleotide to one or more moieties or conjugates toenhance the activity, cellular distribution, or cellular uptake of theantisense oligonucleotide. Such moieties or conjugates include lipidssuch as cholesterol, cholic acid, thioether, aliphatic chains,phospholipids, polyamines, polyethylene glycol (PEG), palmityl moieties,and others as disclosed in, for example, U.S. Pat. Nos. 5,514,758,5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,597,696 and5,958,773.

Chimeric antisense oligonucleotides are also within the scope of theinvention, and can be prepared from the present inventiveoligonucleotides using the methods described in, for example, U.S. Pat.Nos. 5,013,830, 5,149,797, 5,403,711, 5,491,133, 5,565,350, 5,652,355,5,700,922 and 5,958,773.

Preferred antisense oligonucleotides can be selected by routineexperimentation using, for example, assays described in the Examples.Although the inventors are not bound by a particular mechanism ofaction, it is believed that the antisense oligonucleotides achieve aninhibitory effect by binding to a complementary region of the targetpolynucleotide within the cell using Watson-Crick base pairing. Wherethe target polynucleotide is RNA, experimental evidence indicates thatthe RNA component of the hybrid is cleaved by RNase H (Giles et al.,Nuc. Acids Res. 23:954-61, 1995; U.S. Pat. No. 6,001,653). Generally, ahybrid containing 10 base pairs is of sufficient length to serve as asubstrate for RNase H. However, to achieve specificity of binding, it ispreferable to use an antisense molecule of at least 17 nucleotides, as asequence of this length is likely to be unique among human genes.

As disclosed in U.S. Pat. No. 5,998,383, incorporated herein byreference, the oligonucleotide is selected such that the sequenceexhibits suitable energy related characteristics important foroligonucleotide duplex formation with their complementary templates, andshows a low potential for self-dimerization or self-complementation(Anazodo et al., Biochem. Biophys. Res. Commun. 229:305-09, 1996). Thecomputer program OLIGO (Primer Analysis Software, Version 3.4), is usedto determined antisense sequence melting temperature, free energyproperties, and to estimate potential self-dimer formation andself-complimentarity properties. The program allows the determination ofa qualitative estimation of these two parameters (potential self-dimerformation and self-complimentary) and provides an indication of “nopotential” or “some potential” or “essentially complete potential.”Segments of OAS1 polynucleotides are generally selected that haveestimates of no potential in these parameters. However, segments can beused that have “some potential” in one of the categories. A balance ofthe parameters is used in the selection.

In the antisense art a certain degree of routine experimentation isrequired to select optimal antisense molecules for particular targets.To be effective, the antisense molecule preferably is targeted to anaccessible, or exposed, portion of the target RNA molecule. Although insome cases information is available about the structure of target mRNAmolecules, the current approach to inhibition using antisense is viaexperimentation. According to the invention, this experimentation can beperformed routinely by transfecting cells with an antisenseoligonucleotide using methods described in the Examples. mRNA levels inthe cell can be measured routinely in treated and control cells byreverse transcription of the mRNA and assaying the cDNA levels. Thebiological effect can be determined routinely by measuring cell growthor viability as is known in the art.

Measuring the specificity of antisense activity by assaying andanalyzing cDNA levels is an art-recognized method of validatingantisense results. It has been suggested that RNA from treated andcontrol cells should be reverse-transcribed and the resulting cDNApopulations analyzed. (Branch, A. D., T.I.B.S. 23:45-50, 1998.)According to the present invention, cultures of cells are transfectedwith two different antisense oligonucleotides designed to target OAS1.The levels of mRNA corresponding to OAS1 are measured in treated andcontrol cells.

Additional inhibitors include ribozymes, proteins or polypeptides,antibodies or fragments thereof as well as small molecules. Each ofthese OAS1 inhibitors share the common feature in that they reduce theexpression and/or biological activity of OAS1. In addition to theexemplary OAS1 inhibitors disclosed herein, alternative inhibitors maybe obtained through routine experimentation utilizing methodology eitherspecifically disclosed herein or as otherwise readily available to andwithin the expertise of the skilled artisan.

Ribozymes

OAS1 inhibitors may be ribozymes. A ribozyme is an RNA molecule thatspecifically cleaves RNA substrates, such as mRNA, resulting in specificinhibition or interference with cellular gene expression. As usedherein, the term ribozymes includes RNA molecules that contain antisensesequences for specific recognition, and an RNA-cleaving enzymaticactivity. The catalytic strand cleaves a specific site in a target RNAat greater than stoichiometric concentration.

A wide variety of ribozymes may be utilized within the context of thepresent invention, including for example, the hammerhead ribozyme (forexample, as described by Forster and Symons, Cell 48:211-20, 1987;Haseloff and Gerlach, Nature 328:596-600, 1988; Walbot and Bruening,Nature 334:196, 1988; Haseloff and Gerlach, Nature 334:585, 1988); thehairpin ribozyme (for example, as described by Haseloff et al., U.S.Pat. No. 5,254,678, issued Oct. 19, 1993 and Hempel et al., EuropeanPatent Publication No. 0 360 257, published Mar. 26, 1990); andTetrahymena ribosomal RNA-based ribozymes (see Cech et al., U.S. Pat.No. 4,987,071). Ribozymes of the present invention typically consist ofRNA, but may also be composed of DNA, nucleic acid analogs (e.g.,phosphorothioates), or chimerics thereof (e.g., DNA/RNA/RNA).

Ribozymes can be targeted to any RNA transcript and can catalyticallycleave such transcripts (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat.No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and5,093,246 to Cech et al.). According to certain embodiments of theinvention, any such OAS1 mRNA-specific ribozyme, or a nucleic acidencoding such a ribozyme, may be delivered to a host cell to effectinhibition of OAS1 gene expression. Ribozymes and the like may thereforebe delivered to the host cells by DNA encoding the ribozyme linked to aeukaryotic promoter, such as a eukaryotic viral promoter, such that uponintroduction into the nucleus, the ribozyme will be directlytranscribed.

RNAi

The invention also provides for the introduction of RNA with partial orfully double-stranded character into the cell or into the extracellularenvironment. Inhibition is specific to the OAS1 expression in that anucleotide sequence from a portion of the target OAS1 gene is chosen toproduce inhibitory RNA. This process is (1) effective in producinginhibition of gene expression, and (2) specific to the targeted OAS1gene. The procedure may provide partial or complete loss of function forthe target OAS1 gene. A reduction or loss of gene expression in at least99% of targeted cells has been shown using comparable techniques withother target genes. Lower doses of injected material and longer timesafter administration of dsRNA may result in inhibition in a smallerfraction of cells. Quantitation of gene expression in a cell may showsimilar amounts of inhibition at the level of accumulation of targetmRNA or translation of target protein. Methods of preparing and usingRNAi are generally disclosed in U.S. Pat. No. 6,506,559, incorporatedherein by reference.

The RNA may comprise one or more strands of polymerized ribonucleotide;it may include modifications to either the phosphate-sugar backbone orthe nucleoside. The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses of double-stranded material may yield moreeffective inhibition. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition. RNA containing a nucleotide sequence identical to aportion of the OAS1 target gene is preferred for inhibition. RNAsequences with insertions, deletions, and single point mutationsrelative to the target sequence have also been found to be effective forinhibition. Thus, sequence identity may optimized by alignmentalgorithms known in the art and calculating the percent differencebetween the nucleotide sequences. Alternatively, the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcript.

RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region may be used to transcribe the RNA strand (or strands).

For RNAi, the RNA may be directly introduced into the cell (i.e.,intracellularly), or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining RNA. Methods for oral introduction include direct mixing ofRNA with food of the organism, as well as engineered approaches in whicha species that is used as food is engineered to express an RNA, then fedto the organism to be affected. Physical methods of introducing nucleicacids include injection directly into the cell or extracellularinjection into the organism of an RNA solution.

The advantages of the method include the ease of introducingdouble-stranded RNA into cells, the low concentration of RNA which canbe used, the stability of double-stranded RNA, and the effectiveness ofthe inhibition.

Inhibition of gene expression refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from a OAS1 targetgene. Specificity refers to the ability to inhibit the target genewithout manifest effects on other genes of the cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). For RNA-mediated inhibition in a cell line orwhole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereof.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of dsRNA may result in inhibition in asmaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or95% of targeted cells). Quantitation of OAS1 gene expression in a cellmay show similar amounts of inhibition at the level of accumulation ofOAS1 target mRNA or translation of OAS1 target protein. As an example,the efficiency of inhibition may be determined by assessing the amountof gene product in the cell: mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may comprise one or more strands of polymerized ribonucleotide.It may include modifications to either the phosphate-sugar backbone orthe nucleoside. For example, the phosphodiester linkages of natural RNAmay be modified to include at least one of a nitrogen or sulfurheteroatom. Modifications in RNA structure may be tailored to allowspecific genetic inhibition while avoiding a general panic response insome organisms which is generated by dsRNA. Likewise, bases may bemodified to block the activity of adenosine deaminase. RNA may beproduced enzymatically or by partial/total organic synthesis, anymodified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

RNA containing a nucleotide sequences identical to a portion of the OAS1target gene are preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencemay be effective for inhibition. Thus, sequence identity may optimizedby sequence comparison and alignment algorithms known in the art (seeGribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991,and references cited therein) and calculating the percent differencebetween the nucleotide sequences by, for example, the Smith-Watermanalgorithm as implemented in the BESTFIT software program using defaultparameters (e.g., University of Wisconsin Genetic Computing Group).Greater than 90% sequence identity, or even 100% sequence identity,between the inhibitory RNA and the portion of the OAS1 target gene ispreferred. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the OAS1 target gene transcript (e.g., 400 mM NaCl, 46mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16hours; followed by washing). The length of the identical nucleotidesequences may be at least 25, 50, 100, 200, 300 or 400 bases.

100% sequence identity between the RNA and the OAS1 target gene is notrequired to practice the present invention. Thus the methods have theadvantage of being able to tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism, or evolutionarydivergence.

OAS1 RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. The RNAstrands may or may not be polyadenylated; the RNA strands may or may notbe capable of being translated into a polypeptide by a cell'stranslational apparatus. RNA may be chemically or enzymaticallysynthesized by manual or automated reactions. The RNA may be synthesizedby a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,T3, T7, SP6). The use and production of an expression construct areknown in (see WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425,5,712,135, 5,789,214, and 5,804,693; and the references cited therein).If synthesized chemically or by in vitro enzymatic synthesis, the RNAmay be purified prior to introduction into the cell. For example, RNAcan be purified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. The RNA may bedried for storage or dissolved in an aqueous solution. The solution maycontain buffers or salts to promote annealing, and/or stabilization ofthe duplex strands.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing an organism in a solution containing the RNA. Methods for oralintroduction include direct mixing of the RNA with food of the organism,as well as engineered approaches in which a species that is used as foodis engineered to express the RNA, then fed to the organism to beaffected. For example, the RNA may be sprayed onto a plant or a plantmay be genetically engineered to express the RNA in an amount sufficientto kill some or all of a pathogen known to infect the plant. Physicalmethods of introducing nucleic acids, for example, injection directlyinto the cell or extracellular injection into the organism, may also beused. Vascular or extravascular circulation, the blood or lymph system,and the cerebrospinal fluid are sites where the RNA may be introduced. Atransgenic organism that expresses RNA from a recombinant construct maybe produced by introducing the construct into a zygote, an embryonicstem cell, or another multipotent cell derived from the appropriateorganism.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

The present invention may be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples or subjects. Preferredcomponents are the dsRNA and a vehicle that promotes introduction of thedsRNA. Such a kit may also include instructions to allow a user of thekit to practice the invention.

Suitable injection mixes are constructed so animals receive an averageof 0.5×10⁶ to 1.0×10⁶ molecules of RNA. For comparisons of sense,antisense, and dsRNA activities, injections are compared with equalmasses of RNA (i.e., dsRNA at half the molar concentration of the singlestrands). Numbers of molecules injected per adult are given as roughapproximations based on concentration of RNA in the injected material(estimated from ethidium bromide staining) and injection volume(estimated from visible displacement at the site of injection). Avariability of several-fold in injection volume between individualanimals is possible.

Proteins and Polypeptides

In addition to the antisense molecules and ribozymes disclosed herein,OAS1 inhibitors of the present invention also include proteins orpolypeptides that are effective in either reducing OAS1 gene expressionor in decreasing one or more of OAS1's biological activities, includingbut not limited to enzymatic activity; interaction with single strandedRNA, configurations; and binding to other proteins such as Hepatitis Cvirus NS5A or a fragment thereof. A variety of methods are readilyavailable in the art by which the skilled artisan may, through routineexperimentation, rapidly identify such OAS1 inhibitors. The presentinvention is not limited by the following exemplary methodologies.

Literature is available to the skilled artisan that describes methodsfor detecting and analyzing protein-protein interactions. Reviewed inPhizicky et al., Microbiological Reviews 59:94-123, 1995, incorporatedherein by reference. Such methods include, but are not limited tophysical methods such as, e.g., protein affinity chromatography,affinity blotting, immunoprecipitation and cross-linking as well aslibrary-based methods such as, e.g., protein probing, phage display andtwo-hybrid screening. Other methods that may be employed to identifyprotein-protein interactions include genetic methods such as use ofextragenic suppressors, synthetic lethal effects and unlinkednoncomplementation. Exemplary methods are described in further detailbelow.

Inventive OAS1 inhibitors may be identified through biological screeningassays that rely on the direct interaction between the OAS1 protein anda panel or library of potential inhibitor proteins. Biological screeningmethodologies, including the various “n-hybrid technologies,” aredescribed in, for example, Vidal et al., Nucl. Acids Res. 27(4):919-29,1999; Frederickson, R. M., Curr Opin. Biotechnol. 9(1):90-96, 1998;Brachmann et al., Curr Opin. Biotechnol. 8(5):561-68, 1997; and White,M. A., Proc. Natl. Acad. Sci. U.S.A. 93:10001-03, 1996, each of which isincorporated herein by reference.

The two-hybrid screening methodology may be employed to search new orexisting target cDNA libraries for OAS1 binding proteins that haveinhibitory properties. The two-hybrid system is a genetic method thatdetects protein-protein interactions by virtue of increases intranscription of reporter genes. The system relies on the fact thatsite-specific transcriptional activators have a DNA-binding domain and atranscriptional activation domain. The DNA-binding domain targets theactivation domain to the specific genes to be expressed. Because of themodular nature of transcriptional activators, the DNA-binding domain maybe severed covalently from the transcriptional activation domain withoutloss of activity of either domain. Furthermore, these two domains may bebrought into juxtaposition by protein-protein contacts between twoproteins unrelated to the transcriptional machinery. Thus, two hybridsare constructed to create a functional system. The first hybrid, i.e.,the bait, consists of a transcriptional activator DNA-binding domainfused to a protein of interest. The second hybrid, the target, iscreated by the fusion of a transcriptional activation domain with alibrary of proteins or polypeptides. Interaction between the baitprotein and a member of the target library results in the juxtapositionof the DNA-binding domain and the transcriptional activation domain andthe consequent up-regulation of reporter gene expression.

A variety of two-hybrid based systems are available to the skilledartisan that most commonly employ either the yeast Gal4 or E. coli LexADNA-binding domain (BD) and the yeast Gal4 or herpes simplex virus VP16transcriptional activation domain. Chien et al., Proc. Natl. Acad. Sci.U.S.A. 88:9578-82, 1991; Dalton et al., Cell 68:597-612, 1992; Durfee etal., Genes Dev. 7:555-69, 1993; Vojtek et al., Cell 74:205-14, 1993; andZervos et al., Cell 72:223-32, 1993. Commonly used reporter genesinclude the E. coli lacZ gene as well as selectable yeast genes such asHIS3 and LEU2. Fields et al., Nature (London) 340:245-46, 1989; Durfee,T. K., supra; and Zervos, A. S., supra. A wide variety of activationdomain libraries is readily available in the art such that the screeningfor interacting proteins may be performed through routineexperimentation.

Suitable bait proteins for the identification of OAS1 interactingproteins may be designed based on the OAS1 DNA sequence presented hereinas SEQ ID NO:19. Such bait proteins include either the full-length OAS1protein or fragments thereof.

Plasmid vectors, such as, e.g., pBTM116 and pAS2-1, for preparing OAS1bait constructs and target libraries are readily available to theartisan and may be obtained from such commercial sources as, e.g.,Clontech (Palo Alto, Calif.), Invitrogen (Carlsbad, Calif.) andStratagene (La Jolla, Calif.). These plasmid vectors permit the in-framefusion of cDNAs with the DNA-binding domains as LexA or Gal4BD,respectively.

OAS1 inhibitors of the present invention may alternatively be identifiedthrough one of the physical or biochemical methods available in the artfor detecting protein-protein interactions.

Through the protein affinity chromatography methodology, lead compoundsto be tested as potential OAS1 inhibitors may be identified by virtue oftheir specific retention to OAS1 when either covalently ornon-covalently coupled to a solid matrix such as, e.g., Sepharose beads.The preparation of protein affinity columns is described in, forexample, Beeckmans et al., Eur. J. Biochem. 117:527-35, 1981, andFormosa et al., Methods Enzymol. 208:24-45, 1991. Cell lysatescontaining the full complement of cellular proteins may be passedthrough the OAS1 affinity column. Proteins having a high affinity forOAS1 will be specifically retained under low-salt conditions while themajority of cellular proteins will pass through the column. Such highaffinity proteins may be eluted from the immobilized OAS1 underconditions of high-salt, with chaotropic solvents or with sodium dodecylsulfate (SDS). In some embodiments, it may be preferred to radiolabelthe cells prior to preparing the lysate as an aid in identifying theOAS1 specific binding proteins. Methods for radiolabeling mammaliancells are well known in the art and are provided, e.g., in Sopta et al.,J. Biol. Chem. 260:10353-60, 1985.

Suitable OAS1 proteins for affinity chromatography may be fused to aprotein or polypeptide to permit rapid purification on an appropriateaffinity resin. For example, the OAS1 cDNA may be fused to the codingregion for glutathione S-transferase (GST) which facilitates theadsorption of fusion proteins to glutathione-agarose columns. Smith etal., Gene 67:31-40, 1988. Alternatively, fusion proteins may includeprotein A, which can be purified on columns bearing immunoglobulin G;oligohistidine-containing peptides, which can be purified on columnsbearing Ni²⁺; the maltose-binding protein, which can be purified onresins containing amylose; and dihydrofolate reductase, which can bepurified on methotrexate columns. One exemplary tag suitable for thepreparation of OAS1 fusion proteins that is presented herein is theepitope for the influenza virus hemagglutinin (HA) against whichmonoclonal antibodies are readily available and from which antibodies anaffinity column may be prepared.

Proteins that are specifically retained on a OAS1 affinity column may beidentified after subjecting to SDS polyacrylamide gel electrophoresis(SDS-PAGE). Thus, where cells are radiolabeled prior to the preparationof cell lysates and passage through the OAS1 affinity column, proteinshaving high affinity for OAS1 may be detected by autoradiography. Theidentity of OAS1 specific binding proteins may be determined by proteinsequencing techniques that are readily available to the skilled artisan,such as Mathews, C. K. et al., Biochemistry, The Benjamin/CummingsPublishing Company, Inc., 1990, pp. 166-70.

Small Molecules

The present invention also provides small molecule OAS1 inhibitors thatmay be readily identified through routine application of high-throughputscreening (HTS) methodologies. Reviewed by Persidis, A., NatureBiotechnology 16:488-89, 1998. HTS methods generally refer to thosetechnologies that permit the rapid assaying of lead compounds, such assmall molecules, for therapeutic potential. HTS methodology employsrobotic handling of test materials, detection of positive signals andinterpretation of data. Such methodologies include, e.g., roboticscreening technology using soluble molecules as well as cell-basedsystems such as the two-hybrid system described in detail above.

A variety of cell line-based HTS methods are available that benefit fromtheir ease of manipulation and clinical relevance of interactions thatoccur within a cellular context as opposed to in solution. Leadcompounds may be identified via incorporation of radioactivity orthrough optical assays that rely on absorbance, fluorescence orluminescence as read-outs. See, e.g., Gonzalez et al., Curr. Opin.Biotechnol. 9(6):624-31, 1998, incorporated herein by reference.

HTS methodology may be employed, e.g., to screen for lead compounds thatblock one of OAS1's biological activities. By this method, OAS1 proteinmay be immunoprecipitated from cells expressing the protein and appliedto wells on an assay plate suitable for robotic screening. Individualtest compounds may then be contacted with the immunoprecipitated proteinand the effect of each test compound on OAS1.

Methods for Assessing the Efficacy of OAS1 Inhibitors

Lead molecules or compounds, whether antisense molecules or ribozymes,proteins and/or peptides, antibodies and/or antibody fragments or smallmolecules, that are identified either by one of the methods describedherein or via techniques that are otherwise available in the art, may befurther characterized in a variety of in vitro, ex vivo and in vivoanimal model assay systems for their ability to inhibit OAS1 geneexpression or biological activity. As discussed in further detail in theExamples provided below, OAS1 inhibitors of the present invention areeffective in reducing OAS1 expression levels. Thus, the presentinvention further discloses methods that permit the skilled artisan toassess the effect of candidate inhibitors.

Candidate OAS1 inhibitors may be tested by administration to cells thateither express endogenous OAS1 or that are made to express OAS1 bytransfection of a mammalian cell with a recombinant OAS1 plasmidconstruct.

Effective OAS1 inhibitory molecules will be effective in reducing theenzymatic activity of OAS1 or ability of OAS1 to respond to IFNinduction. Methods of measuring OAS1 enzymatic activity and IFNinduction are known in the art, for example, as described in Eskildsenet al., Nuc. Acids Res. 31:3166-3173, 2003; and Justesen et al., Nuc.Acids Res. 8:3073-3085, 1980, incorporated herein by reference. Theeffectiveness of a given candidate antisense molecule may be assessed bycomparison with a control “antisense” molecule known to have nosubstantial effect on OAS1 expression when administered to a mammaliancell.

OAS1 inhibitors effective in reducing OAS1 gene expression by one ormore of the methods discussed above may be further characterized invitro for efficacy in one of the readily available established cellculture or primary cell culture model systems as described herein, inreference to use of Vero cells challenged by infection with aflavivirus, such as dengue virus.

Pharmaceutical Compositions

The antisense oligonucleotides and ribozymes of the present inventioncan be synthesized by any method known in the art for ribonucleic ordeoxyribonucleic nucleotides. For example, the oligonucleotides can beprepared using solid-phase synthesis such as in an Applied Biosystems380B DNA synthesizer. Final purity of the oligonucleotides is determinedas is known in the art.

The antisense oligonucleotides identified using the methods of theinvention modulate tumor cell proliferation. Therefore, pharmaceuticalcompositions and methods are provided for interfering with virusinfection, preferably flavivirus, most preferably HCV infection,comprising contacting tissues or cells with one or more of antisenseoligonucleotides identified using the methods of the invention.

The invention provides pharmaceutical compositions of antisenseoligonucleotides and ribozymes complementary to the OAS1 mRNA genesequence as active ingredients for therapeutic application. Thesecompositions can also be used in the method of the present invention.When required, the compounds are nuclease resistant. In general thepharmaceutical composition for inhibiting virus infection in a mammalincludes an effective amount of at least one antisense oligonucleotideas described above needed for the practice of the invention, or afragment thereof shown to have the same effect, and a pharmaceuticallyphysiologically acceptable carrier or diluent.

The compositions can be administered orally, subcutaneously, orparenterally including intravenous, intraarterial, intramuscular,intraperitoneally, and intranasal administration, as well as intrathecaland infusion techniques as required. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention. Cationic lipids may also be included in the composition tofacilitate oligonucleotide uptake. Implants of the compounds are alsouseful. In general, the pharmaceutical compositions are sterile.

By bioactive (expressible) is meant that the oligonucleotide isbiologically active in the cell when delivered directly to the celland/or is expressed by an appropriate promotor and active when deliveredto the cell in a vector as described below. Nuclease resistance isprovided by any method known in the art that does not substantiallyinterfere with biological activity as described herein.

“Contacting the cell” refers to methods of exposing or delivering to acell antisense oligonucleotides whether directly or by viral ornon-viral vectors and where the antisense oligonucleotide is bioactiveupon delivery.

The nucleotide sequences of the present invention can be deliveredeither directly or with viral or non-viral vectors. When delivereddirectly the sequences are generally rendered nuclease resistant.Alternatively, the sequences can be incorporated into expressioncassettes or constructs such that the sequence is expressed in the cell.Generally, the construct contains the proper regulatory sequence orpromotor to allow the sequence to be expressed in the targeted cell.

Once the oligonucleotide sequences are ready for delivery they can beintroduced into cells as is known in the art. Transfection,electroporation, fusion, liposomes, colloidal polymeric particles, andviral vectors as well as other means known in the art may be used todeliver the oligonucleotide sequences to the cell. The method selectedwill depend at least on the cells to be treated and the location of thecells and will be known to those skilled in the art. Localization can beachieved by liposomes, having specific markers on the surface fordirecting the liposome, by having injection directly into the tissuecontaining the target cells, by having depot associated in spatialproximity with the target cells, specific receptor mediated uptake,viral vectors, or the like.

The present invention provides vectors comprising an expression controlsequence operatively linked to the oligonucleotide sequences of theinvention. The present invention further provides host cells, selectedfrom suitable eukaryotic and prokaryotic cells, which are transformedwith these vectors as necessary.

Vectors are known or can be constructed by those skilled in the art andshould contain all expression elements necessary to achieve the desiredtranscription of the sequences. Other beneficial characteristics canalso be contained within the vectors such as mechanisms for recovery ofthe oligonucleotides in a different form. Phagemids are a specificexample of such beneficial vectors because they can be used either asplasmids or as bacteriophage vectors. Examples of other vectors includeviruses such as bacteriophages, baculoviruses and retroviruses, DNAviruses, liposomes and other recombination vectors. The vectors can alsocontain elements for use in either procaryotic or eucaryotic hostsystems. One of ordinary skill in the art will know which host systemsare compatible with a particular vector.

The vectors can be introduced into cells or tissues by any one of avariety of known methods within the art. Such methods can be foundgenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York, 1989, 1992; in Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md., 1989; Chang et al., Somatic Gene Therapy, CRC Press, AnnArbor, Mich., 1995; Vega et al., Gene Targeting, CRC Press, Ann Arbor,Mich., 1995; Vectors: A Survey of Molecular Cloning Vectors and TheirUses, Butterworths, Boston, Mass., 1988; and Gilboa et al.,BioTechniques 4:504-12, 1986, and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors.

Recombinant methods known in the art can also be used to achieve theantisense inhibition of a target nucleic acid. For example, vectorscontaining antisense nucleic acids can be employed to express anantisense message to reduce the expression of the target nucleic acidand therefore its activity.

The present invention also provides a method of evaluating if a compoundinhibits transcription or translation of an OAS1 gene and therebymodulates (i.e., reduces) the ability of the cell to activate RNaseL,comprising transfecting a cell with an expression vector comprising anucleic acid sequence encoding OAS1, the necessary elements for thetranscription or translation of the nucleic acid; administering a testcompound; and comparing the level of expression of the OAS1 with thelevel obtained with a control in the absence of the test compound.

PREFERRED EMBODIMENTS

Utilizing methods described above and others known in the art, thepresent invention contemplates a screening method comprising treating,under amplification conditions, a sample of genomic DNA, isolated from ahuman, with a PCR primer pair for amplifying a region of human genomicDNA containing any of nucleotide (nt) positions 2135728, 2135749,2135978, 2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587,2144294, 2144985, 2156523, 2156595, or 2156638 of oligoadenylatesynthetase (OAS1, SEQ ID NO:19). Amplification conditions include, in anamount effective for DNA synthesis, the presence of PCR buffer and athermocycling temperature. The PCR product thus produced is assayed forthe presence of a point mutation at the relevant nucleotide position. Inone embodiment, the PCR product contains a continuous nucleotidesequence comprising about 358 base pairs (bp) written from 5′ to 3′direction and including position 2135728 (mutation 1), 2135749 (mutation2), 2135978 (mutation 3), 2144072 (mutation 4), 2144088 (mutation 5),2144116 (mutation 6), or 2144321 (mutation 7) and the approximately 175bases flanking the position at each side. In another embodiment, theamplicons as described above in Tables 1 and 2 are exemplary of the PCRproducts and corresponding primers.

In one preferred embodiment, the PCR product is assayed for thecorresponding mutation by treating the amplification product, underhybridization conditions, with an oligonucleotide probe specific for thecorresponding mutation, and detecting the formation of any hybridizationproduct. Preferred oligonucleotide probes comprise a nucleotide sequenceindicated in Table 3 below. Oligonucleotide hybridization to targetnucleic acid is described in U.S. Pat. No. 4,530,901.

TABLE 3 Mutation SEQ ID NO Probe sequence SEQ ID NO: 1 GTAGATTTTGCC YGAACAGGTCAGT (SEQ ID NO: 12) SEQ ID NO: 2 CAGTTGACTGGC R GCTATAAACCTA(SEQ ID NO: 13) SEQ ID NO: 3 CAGAGGAGGGGT R GGGGGAGGAGA (SEQ ID NO: 14)SEQ ID NO: 4 TCTCACCCTTTCA R GCTGAAAGCAAC (SEQ ID NO: 15) SEQ ID NO: 5GAAAGCAACAGT R CAGACGATGAGA (SEQ ID NO: 16) SEQ ID NO: 6 ACGATCCCAGGA SGTATCAGAAATAT (SEQ ID NO: 17) SEQ ID NO: 7 TTGATCCAGAGA R GACAAAGCTCCTC(SEQ ID NO: 18) Wherein R = A/G, S = C/G, and Y = C/T.

The PCR admixture thus formed is subjected to a plurality of PCRthermocycles to produce OAS1 and OAS1R gene amplification products. Theamplification products are then treated, under hybridization conditions,with an oligonucleotide probe specific for each mutation. Anyhybridization products are then detected.

The following examples are intended to illustrate but are not to beconstrued as limiting of the specification and claims in any way.

EXAMPLES Example 1 Preparation and Preliminary Screening of Genomic DNA

This example relates to screening of DNA from two specific populationsof patients, but is equally applicable to other patient groups in whichrepeated exposure to HCV is documented, wherein the exposure does notresult in infection. The example also relates to screening patients whohave been exposed to other flaviviruses as discussed above, wherein theexposure did not result in infection.

Here, two populations are studied: (1) a hemophiliac population, chosenwith the criteria of moderate to severe hemophilia, and receipt ofconcentrated clotting factor before January, 1987; and (2) anintravenous drug user population, with a history of injection for over10 years, and evidence of other risk behaviors such as sharing needles.The study involves exposed but HCV negative patients, and exposed andHCV positive patients.

High molecular weight DNA is extracted from the white blood cells fromIV drug users, hemophiliac patients, and other populations at risk ofhepatitis C infection, or infection by other flaviviruses. For theinitial screening of genomic DNA, blood is collected after informedconsent from the patients of the groups described above andanticoagulated with a mixture of 0.14M citric acid, 0.2M trisodiumcitrate, and 0.22M dextrose. The anticoagulated blood is centrifuged at800×g for 15 minutes at room temperature and the platelet-rich plasmasupernatant is discarded. The pelleted erythrocytes, mononuclear andpolynuclear cells are resuspended and diluted with a volume equal to thestarting blood volume with chilled 0.14M phosphate buffered saline(PBS), pH 7.4. The peripheral blood white blood cells are recovered fromthe diluted cell suspension by centrifugation on low endotoxinFicoll-Hypaque (Sigma Chem. Corp. St. Louis, Mo.) at 400×g for 10minutes at 18° C. (18° C.). The pelleted white blood cells are thenresuspended and used for the source of high molecular weight DNA.

The high molecular weight DNA is purified from the isolated white bloodcells using methods well known to one skilled in the art and describedby Maniatis, et al., Molecular Cloning: A Laboratory Manual, 2nd ed.Cold Spring Harbor Laboratory, Sections 9.16-9.23, (1989) and U.S. Pat.No. 4,683,195.

Each sample of DNA is then examined for a point mutation of any one ofthe nucleotides at position 2135728, 2135749, 2135978, 2144072, 2144088,2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985, 2156523,2156595, or 2156638 with reference to the nucleotides positions ofGenbank Accession No. NT_(—)009775.13, corresponding to theoligoadenylate synthetase 1 gene (OAS1).

Example 2 Mutations in OAS1 Gene Associated with Resistance to HCVInfection

Using methods described in Example 1, a population of unrelatedhemophiliac patients and intravenous drug users was studied, and thepresence or absence of a mutation in OAS1 as disclosed in SEQ IDNO:1-SEQ ID NO:7 and SEQ ID NO:57-64 was determined.

In a study of 20 cases and 42 controls in a Caucasian population, thesemutations were found in the context of resistance to hepatitis Cinfection. There was a statistically significant correlation betweenresistance to HCV infection and presence of a point mutation in OAS1.

Example 3 Preparation and Sequencing of cDNA

Total cellular RNA is purified from cultured lymphoblasts or fibroblastsfrom the patients having the hepatitis C resistance phenotype. Thepurification procedure is performed as described by Chomczynski, et al.,Anal. Biochem., 162:156-159 (1987). Briefly, the cells are prepared asdescribed in Example 1. The cells are then homogenized in 10 milliliters(ml) of a denaturing solution containing 4.0M guanidine thiocyanate,0.1M Tris-HCl at pH 7.5, and 0.1M beta-mercaptoethanol to form a celllysate. Sodium lauryl sarcosinate is then admixed to a finalconcentration of 0.5% to the cell lysate after which the admixture wascentrifuged at 5000×g for 10 minutes at room temperature. The resultantsupernatant containing the total RNA is layered onto a cushion of 5.7Mcesium chloride and 0.01M EDTA at pH 7.5 and is pelleted bycentrifugation. The resultant RNA pellet is dissolved in a solution of10 mM Tris-HCl at pH 7.6 and 1 mM EDTA (TE) containing 0.1% sodiumdocecyl sulfate (SDS). After phenolchloroform extraction and ethanolprecipitation, the purified total cellular RNA concentration isestimated by measuring the optical density at 260 nm.

Total RNA prepared above is used as a template for cDNA synthesis usingreverse transcriptase for first strand synthesis and PCR witholigonucleotide primers designed so as to amplify the cDNA in twooverlapping fragments designated the 5′ and the 3′ fragment. Theoligonucleotides used in practicing this invention are synthesized on anApplied Biosystems 381A DNA Synthesizer following the manufacturer'sinstructions. PCR is conducted using methods known in the art. PCRamplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in severaltexts including PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, New York (1989); and PCR.Protocols: A Guide to Methods and Applications, Innis, et al., eds.,Academic Press, San Diego, Calif. (1990) and primers as described inTable 1 herein.

The sequences determined directly from the PCR-amplified DNAs from thepatients with and without HCV infection, are analyzed. The presence of amutation upstream from the coding region of the OAS gene can be detectedin patients who are seronegative for HCV despite repeated exposures tothe virus.

Example 4 Preparation of PCR Amplified Genomic DNA Containing a PointMutation and Detection by Allele Specific Oligonucleotide Hybridization

The point mutation in an oligoadenylate synthetase (OAS1) gene at one ofnucleotide positions 2135728, 2135749, 2135978, 2144072, 2144088,2144116, 2144321, 2131025, 2133961, 2139587, 2144294, 2144985, 2156523,2156595, or 2156638 can be determined by an approach in which PCRamplified genomic DNA containing the mutation is detected byhybridization with oligonucleotide probes that hybridized to thatregion. To amplify the region having the point mutation forhybridization with oligonucleotide specific probes, PCR amplificationsare performed as essentially described in Example 3 with, for example,180 ng of each of the primers shown in Table 1.

Following the PCR amplification, 2 μl of the amplified oligoadenylatesynthetase DNA products are spotted onto separate sheets ofnitrocellulose. After the spotted amplified DNA has dried, thenitrocellulose is treated with 0.5N NaOH for 2 minutes, 1M Tris-HCl atpH 7.5 for 2 minutes, followed by 0.5M Tris-HCl at pH 7.5 containing1.5M NaCl for 2 minutes to denature and then neutralize the DNA. Theresultant filters are baked under a vacuum for 1 hour at 80° C., areprehybridized for at least 20 minutes at 42° C. with a prehybridizationsolution consisting of 6×SSC (1×=0.15M NaCl, 0.15M sodium citrate),5×Denhardt's solution (5×=0.1% polyvinylpyrrolidone, 0.1% ficoll, and0.1% bovine serum albumin), 5 mM sodium phosphate buffer at pH 7.0, 0.5mg/ml salmon testis DNA and 1% SDS.

After the prehybridization step, the nitrocellulose filters areseparately exposed to ³²P-labeled oligonucleotide probes diluted inprehybridization buffer. Labeling of the probes with ³²P is performed byadmixing 2.5 μl of 10× concentrate of kinase buffer (10×=0.5MTris[hydroxymethyl]aminomethane hydrochloride (Tris-HCl) at pH 7.6, 0.1MMgCl₂, 50 mM dithiothreitol (DTT), 1 mM spermidine-HCl, and 1 mMethylenediaminetetraacetic acid (EDTA)), 1.1 μl of 60 μg/μl of aselected oligonucleotide, 18.4 μl water, 2 μl of 6000 Ci/mM of gamma ³²PATP at a concentration of 150 mCi/μl, and 1 μl of 10 U/μl polynucleotidekinase. The labeling admixture is maintained for 20 minutes at 37° C.followed by 2 minutes at 68° C. The maintained admixture is then appliedto a Sephadex G50 (Pharmacia, Inc., Piscataway, N.J.) spin column toremove unincorporated ³²P-labeled ATP.

The oligonucleotide probes used to hybridize to the region containingthe mutation are shown in Table 3 above. The underlined nucleotidecorresponds to the mutation nucleotide. In probes for detecting wildtype (normal), the underlined nucleotide is replaced with the wild-typenucleotide.

Ten×10⁶ cpm of the normal and mutant labeled probes are separatelyadmixed with each filter. The nitrocellulose filters are then maintainedovernight at 42° C. to allow for the formation of hybridizationproducts. The nitrocellulose filters exposed to the normal probe arewashed with 6×SSC containing 0.1% SDS at 46° C. whereas the filtersexposed to the mutant probe are washed with the same solution at a morestringent temperature of 52° C. The nitrocellulose filters are thendried and subjected to radioautography.

Only those products having the point mutation hybridize with the mutantprobe. Positive and negative controls are included in each assay todetermine whether the PCR amplification is successful. Thus, thepatients' genomic DNA prepared in Example 1 are determined by thisapproach to have the unique point mutation of a non-wild type nucleotidesubstituted for a wild type nucleotide at the indicated position.

Example 5 Antisense Inhibition of Target RNA A. Preparation ofOligonucleotides for Transfection

A carrier molecule, comprising either a lipitoid or cholesteroid, isprepared for transfection by diluting to 0.5 mM in water, followed bysonication to produce a uniform solution, and filtration through a 0.45μm PVDF membrane. The lipitoid or cholesteroid is then diluted into anappropriate volume of OptiMEM™ (Gibco/BRL) such that the finalconcentration would be approximately 1.5-2 mmol lipitoid per μgoligonucleotide.

Antisense and control oligonucleotides are prepared by first diluting toa working concentration of 100 μM in sterile Millipore water, thendiluting to 2 μM (approximately 20 mg/mL) in OptiMEM™. The dilutedoligonucleotides are then immediately added to the diluted lipitoid andmixed by pipetting up and down.

B. Transfection

Human PH5CH8 hepatocytes, which are susceptible to HCV infection andsupportive of HCV replication, are used (Dansako et al., Virus Res.97:17-30, 2003; Ikeda et al., Virus Res. 56:157-167, 1998; Noguchi andHirohashi, In Vitro Cell Dev. Biol Anim. 32:135-137, 1996.) The cellsare transfected by adding the oligonucleotide/lipitoid mixture,immediately after mixing, to a final concentration of 300 nMoligonucleotide. The cells are then incubated with the transfectionmixture overnight at 37° C., 5% CO₂ and the transfection mixture remainson the cells for 3-4 days.

C. Total RNA Extraction and Reverse Transcription

Total RNA is extracted from the transfected cells using the RNeasy™ kit(Qiagen Corporation, Chatsworth, Calif.), following protocols providedby the manufacturer. Following extraction, the RNA isreverse-transcribed for use as a PCR template. Generally 0.2-1 μg oftotal extracted RNA is placed into a sterile microfuge tube, and wateris added to bring the total volume to 3 μL. 7 μL of a buffer/enzymemixture is added to each tube. The buffer/enzyme mixture is prepared bymixing, in the order listed:

-   -   4 μL 25 mM MgCl₂    -   2 μL 10× reaction buffer    -   8 μL 2.5 mM dNTPs    -   1 μL MuLV reverse transcriptase (50 u) (Applied Biosystems)    -   1 μL RNase inhibitor (20 u)    -   1 μL oligo dT (50 pmol)

The contents of the microfuge tube are mixed by pipetting up and down,and the reaction is incubated for 1 hour at 42° C.

D. PCR Amplification and Quantification of Target Sequences

Following reverse transcription, target genes are amplified using theRoche Light Cycler™ real-time PCR machine. 20 μL aliquots of PCRamplification mixture are prepared by mixing the following components inthe order listed: 2 μL 10×PCR buffer II (containing 10 mM Tris pH 8.3and 50 mM KCl, Perkin-Elmer, Norwalk, Conn.) 3 mM MgCl₂, 140 μM eachdNTP, 0.175 pmol of each OAS1 oligo, 1:50,000 dilution of SYBR® Green,0.25 mg/mL BSA, 1 unit Taq polymerase, and H₂O to 20 μL. SYBR® Green(Molecular Probes, Eugene, Oreg.) is a dye that fluoresces when bound todouble-stranded DNA, allowing the amount of PCR product produced in eachreaction to be measured directly. 2 μL of completed reversetranscription reaction is added to each 20 μL aliquot of PCRamplification mixture, and amplification is carried out according tostandard protocols.

Example 6 Treatment of Cells with OAS1 RNAi

Using the methods of Example 5, for antisense treatment, cells aretreated with an oligonucleotide based on the OAS1 sequence (SEQ IDNO:19). Two complementary ribonucleotide monomers with deoxy-TTextensions at the 3′ end are synthesized and annealed. Cells of thePH3CH8 hepatocyte cell line are treated with 50-200 nM RNAi with 1:3 L2lipitoid. Cells are harvested on day 1, 2, 3 and 4, and analyzed forOAS1 protein by Western analysis, as described by Dansako et al., VirusRes. 97:17-30, 2003.

Example 7 OAS1 Interaction with Hepatitis C Virus NS5A Protein

The ability of an OAS1 protein or polypeptide of the invention tointeract with hepatitis C virus NS5A protein is assayed using a methoddescribed in Taguchi, T. et al., J. Gen. Virol. 85:959-969, 2004.Polynucleotides encoding OAS1 proteins and polypeptides are prepared asdescribed above, and plasmids are constructed using routine methods,such as described in Taguchi, T. et al One plasmid contains apolynucleotide encoding an OAS1 protein or polypeptide, and a secondplasmid contains polynucleotide encoding NS5A. The plasmids also encodeappropriate tags for the respective proteins, such as FLAG-tag, HA, orGST. Suitable cells, such as HeLa cells, are transiently transfectedwith a plasmid encoding a tag and NS5A protein, and a plasmid encoding adifferent tag and an OAS1 protein or polypeptide. After incubation andpreparation of supernatant as described (Taguchi, T. et al.), a varietyof analytic techniques can be used to detect and quantify the binding ofNS5A with the OAS1 protein or polypeptide. Such techniques are known inthe art and include co-precipitation analysis, immunofluorescenceanalysis, and immunoblot analysis. OAS1 proteins and polynucleotidesthat do not exhibit binding to NS5A are appropriate for further analysisas inhibitors of hepatitis C infection.

Example 8 Chemically and Sterically Conserved Regions of OAS1

As one skilled in the art will recognize, when modifying the structureof OAS1 to improve enzymatic activity or therapeutic potential, certainresidues or regions of residues must be chemically and structurallyconserved. By example, several conserved domains are described below. Asone skilled in the art will recognize, chemically conservative changesto some amino acids that preserve the structure and function of theprotein may be tolerated. For example, Asp75 and Asp77 both coordinatecatalytic divalent metal ions that are essential to OAS1 function. Whilemodifications to these bases may be tolerated (e.g. to asparagine orglutamic acid), the essentially polar and acid nature of these residuesmust be preserved.

As examples, with regard to SEQ ID NO: 26-29, SEQ ID NO:33, SEQ IDNO:34, and SEQ ID NO:50, the following polypeptide fragments representconserved domains:

(SEQ ID NO: 75) Amino Acids 40-47: FLKERCFR (SEQ ID NO: 76) Amino acids55-82: VSKVVKGGSSGKGTTLRGRSDADLVVFL (SEQ ID NO: 77) Amino Acids 94-112:RRGEFIQEIRRQLEACQRE (SEQ ID NO: 78) Amino Acids 128-138: NPRALSFVLSS(SEQ ID NO: 79) Amino Acids 145-158: VEFDVLPAFDALGQ (SEQ ID NO: 80)Amino Acids 182-198: KEGEFSTCFTELQRDFL (SEQ ID NO: 81) Amino Acids201-217: RPTKLKSLIRLVKLHWYQ (SEQ ID NO: 82) Amino Acids 225-241:KLPPQYALELLTVYAWE (SEQ ID NO: 83) Amino Acids 296-307: PVILDPADPTGN (SEQID NO: 84) Amino Acids 337-343: GSPVSSW

Example 9 Amino Acids Changes that Improve Enzyme Active Site

Changes in OAS amino acids sequences can be envisioned that improve theenzymatic activity of the protein. In one preferred embodiment, aminoacids within the active site of the enzyme can be modified to improveATP or metal ion binding, enzyme efficiency, and enzyme processivity. Anexample of such an alteration would be the substitution of a tyrosinefor a glycine at amino acid position 61 of SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:48. Substitutionof the chemically innocuous glycine for the polar tyrosine shouldfacilitate hydrogen bonding between the N3 atom of ATP and this aminoacid position, thereby improving the dissociation constant andenergetics of this interaction. A tyrosine is found at this position in,for example, the more processive poly-A polymerase. As one skilled inthe art will recognize, other modifications can be envisioned.

Example 10 Amino Acid Changes that Improve Double-Stranded RNA Binding

A second example of amino acid modifications to OAS that improveenzymatic activity would be those that stabilize the interaction betweenthis protein and double-stranded viral RNA. The table below lists thoseamino acids in the RNA binding groove of the protein and severalproposed changes designed to stabilize the interaction between thebasic, positively charged amino acid side chains and the negativelycharged ribonucleic acid. Changes are envisioned that increase thepositive charge density in the RNA binding groove of the protein. As oneskilled in the art will recognize, similar types of modifications to theRNA binding groove can be envisioned.

TABLE 4 Proposed changes to amino acids in RNA binding groove Amino AcidPosition Proposed Modification Glycine 39 Arginine or Lysine Lysine 42Arginine Lysine 60 Arginine Arginine 195 Lysine Lysine 199 ArginineLysine 204 Arginine

Example 11 Analysis of Genetic Mutations

Those skilled in the art will recognize that numerous other analyticalmethods exist for assessing the evolutionary importance of particularmutations in a genetic analysis. One example is the well-knowncalculation of a linkage disequilibrium estimate, commonly referred toas D′ (Lewontin, Genetics 49:49-67, 1964). Other particularly relevantmethods attempt to estimate selective pressures and/or recentevolutionary events within a genetic locus (for example, selectivesweeps) by comparing the relative abundance of high-, moderate-, orlow-frequency mutations in the locus. Most familiar of these tests isthe Tajima D statistic (Tajima, Genetics 123:585-595, 1989). Fu and L1,Genetics 133:693-709 (1993) have also developed a variant to the Tajimaand other statistics that also makes use of knowledge regarding theancestral allele for each mutation. These and other methods are appliedto the mutations of the present invention to assess relativecontribution to the observed effects.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of theinvention. All patents, patent publications, and non-patent publicationscited are incorporated by reference herein.

1. A human genetic screening method for identifying an oligoadenylatesynthetase gene (OAS1) mutation comprising detecting in a nucleic acidsample the presence of an OAS1 point mutation selected from the groupconsisting of: substitution of a non-reference nucleotide for areference nucleotide at nucleotide position 2135728, 2135749, 2135978,2144072, 2144088, 2144116, 2144321, 2131025, 2133961, 2139587, 2144294,2144985, 2156523, and 2156638 of reference sequence SEQ ID NO:19; anddeletion of the reference nucleotide at position 2156595 of referencesequence SEQ ID NO:19; thereby identifying said mutation.
 2. (canceled)3. An isolated polypeptide consisting of at least one amino acidsequence selected from the group consisting of SEQ ID NO:75-84 andhaving at least 80% sequence similarity to a polypeptide selected fromthe group consisting of SEQ ID NO:20-30, SEQ ID NO:32-35, and SEQ IDNO:46-52.
 4. (canceled)
 5. The polypeptide of claim 2 covalentlyattached to a polypeptide comprising a protein transduction domain. 6.The polypeptide of claim 5 wherein the protein transduction domain iscomprised of a polypeptide selected from the group consisting of SEQ IDNO:85-94.
 7. The polypeptide of claim 5 wherein the protein transductiondomain is comprised of a polypeptide having at least 80% sequencesimilarity to a polypeptide selected from the group consisting of SEQ IDNO:85-94.
 8. The polypeptide of claim 5 wherein the protein transductiondomain differs from a polypeptide selected from the group consisting ofSEQ ID NO:85-94 by the addition or substitution of an arginine, lysine,or histidine.
 9. The polypeptide of claim 2 covalently attached topolyethylene glycol.
 10. The polypeptide of claim 2 encapsulated in aliposome.
 11. The polypeptide of claim 2 covalently attached to anendosome disrupting agent.
 12. The polypeptide of claim 2 noncovalentlyattached to an endosome disrupting agent.
 13. The polypeptide of claim11 wherein the endosome disrupting agent is pH sensitive.
 14. Thepolypeptide of claim 2 covalently conjugated to a sugar moiety. 15.(canceled)
 16. An isolated polypeptide produced by the methodcomprising: (a) expressing the polypeptide of claim 2 by a cell; and (b)recovering said polypeptide.
 17. The polypeptide of claim 2 which isproduced by a recombinant host cell.
 18. The polypeptide of claim 2comprising a heterologous polypeptide sequence.
 19. A compositioncomprising the polypeptide of claim 2 and a pharmaceutically-acceptablecarrier.
 20. An isolated polynucleotide comprising a nucleotide sequencethat encodes the polypeptide sequence of claim
 2. 21. An isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:31, SEQ ID NO:3645 and SEQ ID NO:55-56. 22-81.(canceled)